Sample records for a-ii coagulation factor

Acquired coagulationfactor inhibitors are an autoimmune disease causing bleeding symptoms due to decreases in the corresponding factor (s) which result from the appearance of autoantibodies against coagulationfactors (inhibitor). This disease is quite different from congenital coagulationfactor deficiencies based on genetic abnormalities. In recent years, cases with this disease have been increasing, and most have anti-factor VIII autoantibodies. The breakdown of the immune control mechanism is speculated to cause this disease since it is common in the elderly, but the pathology and pathogenesis are presently unclear. We herein describe the pathology and pathogenesis of factor VIII and factor V inhibitors. Characterization of these inhibitors leads to further analysis of the coagulation process and the activation mechanisms of clotting factors. In the future, with the development of new clotting examination method (s), we anticipate that further novel findings will be obtained in this field through inhibitor analysis. In addition, detailed elucidation of the coagulation inhibitory mechanism possibly leading to hemostatic treatment strategies for acquired coagulationfactor disorders will be developed.

Objective: To provide a comprehensive literature review on roles of coagulationfactor XIII (FXIII) in coagulation, wound healing, neoplasm, bone metabolism, and pregnancy. Data Sources: All articles in PubMed with key words Coagulationfactor XIII, wound, leukemia, tumor, bone, and pregnancy with published date from 2001 to 2016 were included in the study. Frequently cited publications before 2000 were also included. Study Selection: We reviewed the role of FXIII in biologic processes as documented in clinical, animal, and in vitro studies. Results: FXIII, a member of the transglutaminase (TG) family, plays key roles in various biological processes. Besides its well-known function in coagulation, the cross-linking of small molecules catalyzed by FXIII has been found in studies to help promote wound healing, improve bone metabolism, and prevent miscarriages. The study has also shown that FXIII concentration level differs in the blood of patients with leukemia and solid tumors and offers promises as a diagnostic indicator. Conclusions: FXIII has many more biologic functions besides being known as coagulationfactor. The TG activity of FXIII contributes to several processes, including wound healing, bone extracellular matrix stabilization, and the interaction between embryo and decidua of uterus. Further research is needed to elucidate the link between FXIII and leukemia and solid tumors. PMID:28091415

It is unfortunate that we cannot report, in the area of coagulation, advances that have been seen in related fields such as thrombolytic therapy. The reported progress (Gold et al, 1984; Van de Werf et al, 1984) with human recombinant tissue plasminogen activator (Pennica et al, 1983) augers well for the application of recombinant technology to the problems faced by patients with coagulation defects. While plasminogen activator is being assessed in an acute therapeutic setting, its use signals a beginning of the application of the technology to abnormalities of the haemostatic mechanism. Chronic administration of coagulationfactors for prophylaxis and replacement therapy would appear to be just one more step down the pathway illuminated by the biochemists, microbiologists and cell biologists who have preceded the clinicians in this promising area. There is no record of the use of genetically engineered materials in the treatment of coagulation defects, primarily because the body of knowledge and refined techniques have only recently been acquired. For this reason we have had to project developments in other areas onto the problems that exist for the haemostatically compromised patient. In describing the potential usefulness of these technologies, it is difficult to ascertain where the logical projection, from a fully investigated model system, diverges from flights of imaginative fancy. Cloning projects considered overly ambitious and grandiose at the beginning of this decade are already accomplished feats. The feasibility of gene therapy in the mammalian system has been demonstrated, and trade publications now discuss governmental approval for investigative use of this procedure in 1985. Panels of physicians, scientists and even politicians now seriously contemplate and promulgate views and regulations pertaining to the efficacy and ethics of the use of genetic engineering in the treatment of human disease. The haemophilias will certainly be among the first

We report on a 12-month-old Japanese boy with an interstitial deletion of the long-arm of chromosome 1 and meningomyelocele, hydrocephalus, anal atresia, atrial septal defect, left renal agenesis, bilateral cryptorchidism, talipes equinovarus, low birth weight, growth/developmental retardation, and many minor anomalies. By conventional GTG-banding, his karyotype was first interpreted as 46,XY,de1(1)(q23q24), but it was corrected as 46,XY.ish del(1)(q24q25.3) by fluorescence in situ hybridization using 11 known cosmid clones as probes. His serum levels of apolipoprotein A-II (gene symbol: APOA2, previously assigned to 1q21-q23) and coagulationfactor V (F5, 1q21-q25) were normal, while serum concentration and activity of antithrombin III (AT3, 1q23-q25.1) was low. The results indicated that localization of APOA2 and F5 are proximal to the deleted region and AT3 is located within the deletion extent in the patient. 16 refs., 4 figs.

Coagulationfactor VIIa (FVIIa) is present at subnanomolar concentration and represents a small percentage of the total amount of FVII in the circulation. FVIIa is poised to initiate blood clotting when it encounters its pivotal cofactor tissue factor (TF) which becomes exposed to blood upon vascular rupture. The requirement for complex formation with TF in order for FVIIa to express procoagulant activity ensures thrombin and fibrin generation at the right time and place. Thus TF acts as a guardian of safety of paramount importance to blood coagulation by providing localization to the site of injury and at the same time inducing maturation of zymogen-like free FVIIa to the active cofactor-bound enzyme. This review gives an account of the accumulated knowledge about the structure, function and TF dependence of FVIIa to arrive at a plausible allosteric mechanism by which TF induces maturation of the active conformation of FVIIa.

Tissue factor (TF) is an integral membrane protein, normally separated from the blood by the vascular endothelium, which plays a key role in the initiation of blood coagulation. With a perforating vascular injury, TF becomes exposed to blood and binds plasma factor VIIa. The resulting complex initiates a series of enzymatic reactions leading to clot formation and vascular sealing. In some pathologic states, circulating blood cells express TF as a result of exposure to an inflammatory stimulus leading to intravascular clotting, vessel occlusion and thrombotic pathology. Numerous controversies have arisen related to the influence of structural features of TF, its presentation and its function. There are contradictory reports about the synthesis and presentation of TF on blood cells and the presence (or absence) of functionally active TF circulating in normal blood either on microparticles or as a soluble protein. In this review we discuss TF structure-function relationships and the role of TF during various phases of the blood coagulation process. We also highlight controversies concerning the expression/presence of TF on various cells and in blood in normal and pathologic states. PMID:19592470

Formation of thrombin is triggered when membrane-localized tissue factor (TF) is exposed to blood. In closed models of this process, thrombin formation displays an initiation phase (low rates of thrombin production cause platelet activation and fibrinogen clotting), a propagation phase (>95% of thrombin production occurs) and a termination phase (prothrombin activation ceases and free thrombin is inactivated). A current controversy centers on whether the TF stimulus requires supplementation from a circulating pool of blood TF in order to sustain an adequate procoagulant response. We have evaluated the requirement for TF during the progress of the blood coagulation reaction and have extended these analyses to assess the requirement for TF during resupply (“flow replacement”). Elimination of TF activity at various times during the initiation phase indicated: a period of absolute dependence (<10s); a transitional period in which the dependence on TF is partial and decreases as the reaction proceeds (10–240s); and a period in which the progress of the reaction is TF independent (>240s). Resupply of reactions late during the termination phase with fresh reactants, but no TF, yielded immediate bursts of thrombin formation similar in magnitude to the original propagation phases. Our data show that independence from the initial TF stimulus is achieved by the onset of the propagation phase and that the ensemble of coagulation products and intermediates which yield this TF independence maintain their prothrombin-activating potential for considerable time. These observations support the hypothesis that the transient, localized expression of TF is sufficient to sustain a TF-independent procoagulant response as long as flow persists. PMID:16215234

Formation of thrombin is triggered when membrane-localized tissue factor (TF) is exposed to blood. In closed models of this process, thrombin formation displays an initiation phase (low rates of thrombin production cause platelet activation and fibrinogen clotting), a propagation phase (>95% of thrombin production occurs), and a termination phase (prothrombin activation ceases and free thrombin is inactivated). A current controversy centers on whether the TF stimulus requires supplementation from a circulating pool of blood TF to sustain an adequate procoagulant response. We have evaluated the requirement for TF during the progress of the blood coagulation reaction and have extended these analyses to assess the requirement for TF during resupply ("flow replacement"). Elimination of TF activity at various times during the initiation phase indicated: a period of absolute dependence (<10 s); a transitional period in which the dependence on TF is partial and decreases as the reaction proceeds (10-240 s); and a period in which the progress of the reaction is TF independent (>240 s). Resupply of reactions late during the termination phase with fresh reactants, but no TF, yielded immediate bursts of thrombin formation similar in magnitude to the original propagation phases. Our data show that independence from the initial TF stimulus is achieved by the onset of the propagation phase and that the ensemble of coagulation products and intermediates that yield this TF independence maintain their prothrombin activating potential for considerable time. These observations support the hypothesis that the transient, localized expression of TF is sufficient to sustain a TF-independent procoagulant response as long as flow persists.

This paper reports an asymptomatic coagulation defect responsible for an abnormality at the contact phase of blood coagulation in vitro, distinct from Hageman factor and Fletcher factor deficiencies. Coagulation studies in a 50-yr-old French woman without bleeding tendency revealed the following results: whole-blood clotting time in glass tubes and activated partial thromboplastin time with kaolin and ellagic acid were greatly prolonged; one-stage prothrombin was normal; no circulating anticoagulant was detected, and the infusion of normal plasma corrected the coagulation defect with an estimated half-life of 6.5 days; the levels of factor VIII, IX, XI, and XII were normal; mutual correction was obtained with a Fletcher factor-deficient plasma; the level of whole complement was normal. Studies of the contact phase of blood coagulation and contact-induced fibrinolysis showed the same abnormalities as in Hageman factor- and Fletcher-deficient plasmas. These results indicate that the patient's plasma is deficient in a previously undescribed coagulationfactor, which participates in the initial stage of the blood coagulation process in vitro. Family studies revealed consanguinity in the propositus' parents. The assay of this newly described factor in the propositus' children revealed a partial defect, compatible with a heterozygous state, in three of the four tested children. This indicates a recessive inheritance of this new blood coagulation defect.

It is not known whether the mammalian mechanism of coagulation initiation is conserved in fish. Identification of factor VII is critical in providing evidence for such a mechanism. A cDNA was cloned from a zebrafish (teleost) library that predicted a protein with sequence similarity to human factor VII. Factor VII was shown to be present in zebrafish blood and liver by Western blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thrombin generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered thrombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and humans and add credence to the zebrafish as a model for mammalian hemostasis. The structure of zebrafish factor VIIa predicted by homology modeling was consistent with the overall three-dimensional structure of human factor VIIa. However, amino acid disparities were found in the epidermal growth factor-2/serine protease regions that are present in the human tissue factor–factor VIIa contact surface, suggesting a structural basis for the species specificity of this interaction. In addition, zebrafish factor VII demonstrates that the Gla-EGF-EGF-SP domain structure, which is common to coagulationfactors VII, IX, X, and protein C, was present before the radiation of the teleosts from the tetrapods. Identification of zebrafish factor VII significantly narrows the evolutionary window for development of the vertebrate coagulation cascade and provides insight into the structural basis for species specificity in the tissue factor–factor VIIa interaction. PMID:11459993

This chapter reports on the efforts of the USEPA to study conventional and enhanced coagulation for the control of disinfection by-products (DBPs) in drinking water. It examines the control of DBPs like trihalomethanes, haloacetic acids and the surrogate total organic halide in t...

Summary Tissue factor (TF) is a transmembrane glycoprotein and an essential component of factor VIIa-TF enzymatic complex that triggers activation of the coagulation cascade. Formation of TF-FVIIa complexes on cell surfaces not only trigger the coagulation cascade but also transduce cell signaling via activation of protease-activated receptors. Tissue factor is expressed constitutively on cell surfaces of a variety of extravascular cell types, including fibroblasts and pericytes in and surrounding blood vessel walls and epithelial cells but generally absent on cells that come in contact with blood directly. However, TF expression could be induced in some blood cells, such as monocytes and endothelial cells, following an injury or pathological stimuli. Tissue factor is essential for hemostasis, but aberrant expression of TF leads to thrombosis. Therefore, a proper regulation of TF activity is critical for the maintenance of hemostatic balance and health in general. TF-FVIIa coagulant activity at the cell surface is influenced not only by TF protein expression levels but also independently by a variety of mechanisms, including alterations in membrane phospholipid composition and cholesterol content, thiol-dependent modifications of TF allosteric disulfide bond, and other post-translational modifications of TF. In this article, we critically review key literature on mechanisms by which TF coagulant activity is regulated at the cell surface in the absence of changes in TF protein levels with specific emphasis on recently published data and provide the authors’ perspective on the subject. PMID:23006890

Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the {gamma}-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced.

Blood coagulation is thought to be initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. In contrast, coagulationfactor XII (FXII)–mediated fibrin formation is not believed to play an important role for coagulation in vivo. We used FXII-deficient mice to study the contributions of FXII to thrombus formation in vivo. Intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in the formation and stabilization of platelet-rich occlusive thrombi. Although FXII-deficient mice do not experience spontaneous or excessive injury-related bleeding, they are protected against collagen- and epinephrine-induced thromboembolism. Infusion of human FXII into FXII-null mice restored injury-induced thrombus formation. These unexpected findings change the long-standing concept that the FXII-induced intrinsic coagulation pathway is not important for clotting in vivo. The results establish FXII as essential for thrombus formation, and identify FXII as a novel target for antithrombotic therapy. PMID:16009717

Epidemiological and experimental data have indicated the beneficial and adverse effects of estrogenic replacement therapy. In the present study, we explored the effect of ethinylestradiol (EE) and 17β-estradiol (E2) on screening tests, prothrombin time (PT) and activated partial thromboplastin time (APTT), as well as the activity of coagulationfactors (FVII, FX, FXI, and FXII) in male Wistar rats. Animals were injected subcutaneously during three consecutive days with EE or E2 (1, 3, 10, and 30 mg/kg) and propylene glycol (0.3 ml; vehicle, V). EE produced significant increments (P<0.05) on PT (8, 13, 15, and 10%) and APTT (32, 35, and 28%), whereas E2 did not show any effect. EE diminished the activity of factors VII (−10, −13, and −10%) and X (−10, −9, −15, and −14%; P<0.05), and E2 (1 mg/kg) produced a modest increment (8%; P<0.05) on FX only. E2 (10 mg/kg) showed a diminution of 9% (P<0.05), while EE did not produce any response on factor XII. EE diminished (−15, −14, −19, and −17%) but E2 augmented (10, 14, 24, and 24%) factor XI activity (P<0.05). Our findings suggest that EE and E2 produce different effects on coagulation and that EE seems to act across an inhibitory mechanism of coagulationfactor activity in the present experimental model. PMID:27829580

Epidemiological and experimental data have indicated the beneficial and adverse effects of estrogenic replacement therapy. In the present study, we explored the effect of ethinylestradiol (EE) and 17β-estradiol (E2) on screening tests, prothrombin time (PT) and activated partial thromboplastin time (APTT), as well as the activity of coagulationfactors (FVII, FX, FXI, and FXII) in male Wistar rats. Animals were injected subcutaneously during three consecutive days with EE or E2 (1, 3, 10, and 30 mg/kg) and propylene glycol (0.3 ml; vehicle, V). EE produced significant increments (P<0.05) on PT (8, 13, 15, and 10%) and APTT (32, 35, and 28%), whereas E2 did not show any effect. EE diminished the activity of factors VII (-10, -13, and -10%) and X (-10, -9, -15, and -14%; P<0.05), and E2 (1 mg/kg) produced a modest increment (8%; P<0.05) on FX only. E2 (10 mg/kg) showed a diminution of 9% (P<0.05), while EE did not produce any response on factor XII. EE diminished (-15, -14, -19, and -17%) but E2 augmented (10, 14, 24, and 24%) factor XI activity (P<0.05). Our findings suggest that EE and E2 produce different effects on coagulation and that EE seems to act across an inhibitory mechanism of coagulationfactor activity in the present experimental model.

We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity. Images PMID:6421875

Studies with riboflavin in the 1960s showed that it could be effective at inactivating pathogens when exposed to light. The principal mode of action is through electron transfer reactions, most importantly in nucleic acids. This suggested that it could act as a photosensitizer useful in the inactivation of pathogens found in blood products. Objective To study the influence of photo-inactivation with riboflavin on the coagulationfactors of plasma. Methods The photo-inactivation procedure of riboflavin plus light was applied. Fifty isogroup pools of two plasmas were made from 100 U of plasma that were derived from whole blood products that had previously been held overnight. Pools were split into two bags. One of them was photo-inactivated, and post inactivation samples were obtained. The second bag was not photo-inactivated and samples were taken. Total protein, fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, antithrombin III, PC, PS, α-2 antiplasmin and vWF:Ag, the multimeric structure of vWF and ADAMTS-13 were analyzed. Results In plasma, the proteins most sensitive to photo-inactivation were fibrinogen, FXI, FVIII, FV, and FIX (33%, 32%, 30%, 18% and 18% loss, respectively). Coagulation inhibitors, PS, antithrombin III and PC showed little decrease (all 2%). Retention of vWF and ADAMTS-13 were 99% and 88%, respectively. Conclusions As with other pathogen reduction procedures for plasma products, treatment with riboflavin and UV light resulted in reduction in the activity levels of several pro-coagulantfactors. Coagulation inhibitors are well preserved. PMID:19782644

To accurately estimate longitudinal changes in individuals, it is important to take into consideration the biological variability of the measurement. The few studies available on the biological variations of coagulation parameters are mostly outdated. We confirmed the published results using modern, fully automated methods. Furthermore, we added data for additional coagulation parameters. At 8:00 am, 12:00 pm, and 4:00 pm on days 1, 3, and 5, venous blood was collected from 31 healthy volunteers. A total of 16 parameters related to coagulation screening tests as well as the activity of coagulationfactors were analyzed; these included prothrombin time, fibrinogen (Fbg), activated partial thromboplastin time, thrombin time, international normalized ratio, prothrombin time activity, activated partial thromboplastin time ratio, fibrin(-ogen) degradation products, as well as the activity of factor II, factor V, factor VII, factor VIII, factor IX, and factor X. All intraindividual coefficients of variation (CVI) values for the parameters of the screening tests (except Fbg) were less than 5%. Conversely, the CVI values for the activity of coagulationfactors were all greater than 5%. In addition, we calculated the reference change value to determine whether a significant difference exists between two test results from the same individual.

Cardiovascular diseases are still the most important cause of morbidity and mortality in western countries and antithrombotic treatment is nowadays widely used. Drugs able to reduce coagulation activation are the treatment of choice for a number of arterial and/or venous thromboembolic conditions. Some of the drugs currently used for this purpose, such as heparins (UFH or LMWH) and VKA, have limitations consisting of a narrow therapeutic window and an unpredictable response with the need of laboratory monitoring in order to assess their efficacy and safety. These drawbacks have stimulated an active research aimed to develop new drugs able to act on single factors involved in the coagulation network, with predictable response. Intense experimental and clinical work on new drugs has focused on synthetic agents, which could preferably be administered orally and at fixed doses. The most advanced clinical development with new anticoagulants has been achieved for those inhibiting FXa and some of them, like fondaparinux, are already currently used in clinical practice. Other agents, such as rivaroxaban, apixaban, otamixaban and edoxaban are under development and have already been studied or are currently under investigation in large scale phase III clinical trials for prevention and treatment of venous thromboembolism, atrial fibrillation and acute coronary syndromes. Some of them have proved to be more effective than conventional therapy. Data on some agents inhibiting FVa are still preliminary and some of these drugs have so far been considered only in patients with disseminated intravascular coagulation secondary to sepsis. PMID:21545479

To assess whether, as has been hypothesised since medieval times, acute fear can curdle blood. Crossover trial. Main meeting room of Leiden University's Department of Clinical Epidemiology, the Netherlands, converted to a makeshift cinema. 24 healthy volunteers aged ≤30 years recruited among students, alumni, and employees of the Leiden University Medical Center: 14 were assigned to watch a frightening (horror) movie followed by a non-threatening (educational) movie and 10 to watch the movies in reverse order. The movies were viewed more than a week apart at the same time of day and both lasted approximately 90 minutes. The primary outcome measures were markers, or "fear factors" of coagulation activity: blood coagulantfactor VIII, D-dimer, thrombin-antithrombin complexes, and prothrombin fragments 1+2. The secondary outcome was participant reported fear experienced during each movie using a visual analogue fear scale. All participants completed the study. The horror movie was perceived to be more frightening than the educational movie on a visual analogue fear scale (mean difference 5.4, 95% confidence interval 4.7 to 6.1). The difference in factor VIII levels before and after watching the movies was higher for the horror movie than for the educational movie (mean difference of differences 11.1 IU/dL (111 IU/L), 95% confidence interval 1.2 to 21.0 IU/dL). The effect of either movie on levels of thrombin-antithrombin complexes, D-dimer, and prothrombin fragments 1+2 did not differ. Frightening (in this case, horror) movies are associated with an increase of blood coagulantfactor VIII without actual thrombin formation in young and healthy adults. Trial registration ClinicalTrials.gov NCT02601053. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Objective To assess whether, as has been hypothesised since medieval times, acute fear can curdle blood. Design Crossover trial. Setting Main meeting room of Leiden University’s Department of Clinical Epidemiology, the Netherlands, converted to a makeshift cinema. Participants 24 healthy volunteers aged ≤30 years recruited among students, alumni, and employees of the Leiden University Medical Center: 14 were assigned to watch a frightening (horror) movie followed by a non-threatening (educational) movie and 10 to watch the movies in reverse order. The movies were viewed more than a week apart at the same time of day and both lasted approximately 90 minutes. Main outcome measures The primary outcome measures were markers, or “fear factors” of coagulation activity: blood coagulantfactor VIII, D-dimer, thrombin-antithrombin complexes, and prothrombin fragments 1+2. The secondary outcome was participant reported fear experienced during each movie using a visual analogue fear scale. Results All participants completed the study. The horror movie was perceived to be more frightening than the educational movie on a visual analogue fear scale (mean difference 5.4, 95% confidence interval 4.7 to 6.1). The difference in factor VIII levels before and after watching the movies was higher for the horror movie than for the educational movie (mean difference of differences 11.1 IU/dL (111 IU/L), 95% confidence interval 1.2 to 21.0 IU/dL). The effect of either movie on levels of thrombin-antithrombin complexes, D-dimer, and prothrombin fragments 1+2 did not differ. Conclusion Frightening (in this case, horror) movies are associated with an increase of blood coagulantfactor VIII without actual thrombin formation in young and healthy adults. Trial registration ClinicalTrials.gov NCT02601053. PMID:26673787

Background and Purpose— Abnormal coagulation has been implicated in the pathogenesis of ischemic stroke, but how this association is mediated and whether it differs between ischemic stroke subtypes is unknown. We determined the shared genetic risk between 14 coagulationfactors and ischemic stroke and its subtypes. Methods— Using genome-wide association study results for 14 coagulationfactors from the population-based TwinsUK sample (N≈2000 for each factor), meta-analysis results from the METASTROKE consortium ischemic stroke genome-wide association study (12 389 cases, 62 004 controls), and genotype data for 9520 individuals from the WTCCC2 ischemic stroke study (3548 cases, 5972 controls—the largest METASTROKE subsample), we explored shared genetic risk for coagulation and stroke. We performed three analyses: (1) a test for excess concordance (or discordance) in single nucleotide polymorphism effect direction across coagulation and stroke, (2) an estimation of the joint effect of multiple coagulation-associated single nucleotide polymorphisms in stroke, and (3) an evaluation of common genetic risk between coagulation and stroke. Results— One coagulationfactor, factor XIII subunit B (FXIIIB), showed consistent effects in the concordance analysis, the estimation of polygenic risk, and the validation with genotype data, with associations specific to the cardioembolic stroke subtype. Effect directions for FXIIIB-associated single nucleotide polymorphisms were significantly discordant with cardioembolic disease (smallest P=5.7×10−04); the joint effect of FXIIIB-associated single nucleotide polymorphisms was significantly predictive of ischemic stroke (smallest P=1.8×10−04) and the cardioembolic subtype (smallest P=1.7×10−04). We found substantial negative genetic covariation between FXIIIB and ischemic stroke (rG=−0.71, P=0.01) and the cardioembolic subtype (rG=−0.80, P=0.03). Conclusions— Genetic markers associated with low FXIIIB levels

Hemorrhage is a major cause of trauma deaths. Coagulopathy exacerbates hemorrhage and is commonly seen during major trauma resuscitation, suggesting that current practice of coagulationfactor transfusion is inadequate. Reversal of coagulopathy involves normalization of body temperature, elimination of the causes of disseminated intravascular coagulation (DIC), and transfusion with fresh-frozen plasma (FFP), platelets, and cryoprecipitate. Transfusion should be guided by clinical factors and laboratory results. However, in major trauma, clinical signs may be obscured and various factors conspire to make it difficult to provide the best transfusion therapy. Existing empiric transfusion strategies for, and prevailing teachings on, FFP transfusion appear to be based on old studies involving elective patients transfused with whole blood and may not be applicable to trauma patients in the era of transfusion with packed red blood cells (PRBCs). Perpetuation of such concepts is in part responsible for the common finding of refractory coagulopathy in major trauma patients today. In this review, we argue that coagulopathy can best be avoided or reversed when severe trauma victims are transfused with at least the equivalent of whole blood in a timely fashion.

The use of international biological standards during the last 30 years has proved extremely successful in promoting global harmonization of estimates between laboratories and methods. Experience has led to the identification of physical criteria essential for standards to be suitable for long-term use. High precision of liquid filling coupled with low residual moisture and oxygen and the use of sealed glass ampoules have been found consistent with homogeneous and stable International Standards (ISs). Most plasma coagulationfactors and inhibitors are calibrated in International Units (IU), which are defined as the amount of analyte in 1 mL of normal pooled plasma. Adoption of the IU has provided clarity in the definition of normal and abnormal states and has facilitated dose calculation for replacement therapy. The assay of like-versus-like materials (e.g., concentrate versus concentrate) has been found to improve interlaboratory agreement and there are now both plasma and concentrate ISs available for many coagulationfactors and inhibitors. Studies into the assay of recombinant factor VIII have indicated that additional measures, such as modifications to assay methodology, are necessary to reduce interlaboratory variability. This experience may prove valuable in the future, when we have to deal increasingly with the challenges to standardization associated with the products of bioengineering.

Coagulation management by transfusion of allogenic blood products and coagulationfactors are competing concepts in current trauma care. Rapid and adequate therapy of trauma-associated coagulopathy is crucial to survival of severely injured patients. Standard coagulation tests such as prothrombin time and activated partial thromboplastin time are commonly used, but these tests are inappropriate for monitoring and guiding therapy in trauma patients. Coagulationfactor-based treatment showed promising results, but randomized trials have not yet been performed. In addition, viscoelastic tests are needed to guide therapy, although there is in fact limited evidence for these in tests in trauma care. Regarding transfusion therapy with allogenic blood products, plasma transfusion has been associated with improved survival in trauma patients following massive transfusion. In contrast, patients not requiring massive transfusion seem to be at risk for suffering complications with increasing volumes of plasma transfused. The collective of trauma patients is heterogeneous. Despite the lack of evidence, there are strong arguments for individualized patient treatment with coagulationfactors for some indications and to abstain from the use of fresh frozen plasma. In patients with severe trauma and major bleeding, plasma, platelets, and red blood cells should be considered to be administered at a ratio of 1 : 1 : 1.

An important function of the liver is the synthesis and secretion of blood coagulationfactors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulationfactors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulationfactors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulationfactors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulationfactor deficiencies.

Plasminogen activators provide effective treatment for patients with acute myocardial infarction. However, paradoxical elevation of thrombin activity associated with failure of clot lysis and recurrent thrombosis has been reported. Generation of thrombin in these circumstances appears to be owing to plasmin (Plm)-induced activation of factor (F) XII. Plm catalyzes proteolysis of several coagulantfactors, but the roles of these factors on Plm-mediated procoagulant activity remain to be determined. Recently developed global coagulation assays were used in this investigation. Rotational thromboelastometry using whole blood, clot waveform analysis and thrombin generation tests using plasma, showed that Plm (> or =125 nmol/l) shortened the clotting times in similar dose-dependent manners. In particular, the thrombin generation test, which was unaffected by products of fibrinolysis, revealed the enhanced coagulation with an approximately two-fold increase of peak level of thrombin generation. Studies using alpha2-antiplasmin-deficient plasma revealed that much lower dose of Plm (> or =16 nmol/l) actually contributed to enhancing thrombin generation. The shortening of clotting time could be observed even in the presence of corn trypsin inhibitor, supporting that Plm exerted the procoagulant activity independently of FXII. In addition, using specific coagulation-deficient plasmas, the clot waveform analysis showed that Plm did not shorten the clotting time in only FV-deficient or FVIII-deficient plasma in prothrombin time-based or activated partial thromboplastin time-based assay, respectively. Our results indicated that Plm did possess procoagulant activity in the blood coagulation, and this effect was likely attributed by multicoagulation factors, dependent on FV and/or FVIII.

Different types of fresh-frozen plasma (FFP) exist, and the concentrations of plasma proteins vary between individuals and blood groups. Furthermore, processing may also influence the content. Quarantine-stored plasma (qFFP) and plasma that was pathogen-reduced using blood-safety (Intercept) technology (piFFP) were analyzed regarding procoagulant and anticoagulant hemostasis proteins, including endogenous thrombin (thrombin-generation) potential (ETP). Thirty-five samples of each type of FFP were analyzed using only male Blood Group O donors. FFP units were stored frozen for comparable periods of time before plasma protein content was assessed. Once the units were thawed, all tests were completed within 4 hours. The results are presented as means ± standard deviations or as median (minimum; maximum) and were compared using independent-sample t tests (significance, p Factor (F)V, FVIII, FXIII, protein S, protein S activity, antithrombin, microvesicle (<900 nm), and α2 antiplasmin were observed in qFFP. The variability of factors was significantly lower in piFFP. Tissue factor (TF) at 1 picomolar (pM) exhibited significantly longer lag time, a lower peak, lower ETP, and a lower velocity index in qFFP compared with piFFP. In TF at 5 pM, significant differences in lag time (longer in qFFP), velocity index (lower in qFFP), and peak (lower in qFFP) were observed. Rotational thromboelastometry revealed a significantly longer (p = 0.002) clot-formation time with intrinsic thromboelastometry for piFFP and a significantly shorter clotting time (p = 0.004) with thromboelastometry fibrinogen testing for piFFP. Pathogen reduction reduces procoagulant and anticoagulant coagulationfactors as well as variability. A thrombin-generation assay showed no reduced ETP and no supraphysiological thrombin generation. None of the

Thrombosis significantly contributes to cancer morbidity and mortality. The mechanism behind thrombosis in cancer may be circulating tissue factor (TF), as levels of circulating TF are associated with thrombosis. However, circulating TF antigen level alone has failed to predict thrombosis in patients with cancer. We hypothesize that coagulationfactor levels regulate the kinetics of circulating TF-induced thrombosis. Coagulation kinetics were measured as a function of individual coagulationfactor levels and TF particle concentration. Clotting times increased when pooled plasma was mixed at or above a ratio of 4:6 with PBS. Clotting times increased when pooled plasma was mixed at or above a ratio of 8:2 with factor VII-depleted plasma, 7:3 with factor IX- or factor X-depleted plasmas, or 2:8 with factor II-, V- or VIII-depleted plasmas. Addition of coagulationfactors VII, X, IX, V and II to depleted plasmas shortened clotting and enzyme initiation times, and increased enzyme generation rates in a concentration-dependent manner. Only additions of factors IX and X from low-normal to high-normal levels shortened clotting times and increased enzyme generation rates. Our results demonstrate that coagulation kinetics for TF particles are controlled by factor IX and X levels within the normal physiological range. We hypothesize that individual patient factor IX and X levels may be prognostic for susceptibility to circulating TF-induced thrombosis.

Background Blood coagulation is a complex network of biochemical reactions, which is peculiar in that it is time- and space-dependent, and has to function in the presence of rapid flow. Recent experimental reports suggest that flow plays a significant role in its regulation. The objective of this study was to use systems biology techniques to investigate this regulation and to identify mechanisms creating a flow-dependent switch in the coagulation onset. Results Using a detailed mechanism-driven model of tissue factor (TF)-initiated thrombus formation in a two-dimensional channel we demonstrate that blood flow can regulate clotting onset in the model in a threshold-like manner, in agreement with existing experimental evidence. Sensitivity analysis reveals that this is achieved due to a combination of the positive feedback of TF-bound factor VII activation by activated factor X (Xa) and effective removal of factor Xa by flow from the activating patch depriving the feedback of "ignition". The level of this trigger (i.e. coagulation sensitivity to flow) is controlled by the activity of tissue factor pathway inhibitor. Conclusions This mechanism explains the difference between red and white thrombi observed in vivo at different shear rates. It can be speculated that this is a special switch protecting vascular system from uncontrolled formation and spreading of active coagulationfactors in vessels with rapidly flowing blood. PMID:20102623

Initiation of blood coagulation by tissue factor (TF) is a robust, highly regulated process. Both the spatial distribution of TF and the geometry of the vasculature may play important roles in regulating coagulation. As this review describes, microfluidic systems provide a unique opportunity for investigating the spatiotemporal dynamics of blood coagulation in vitro. Microfluidic systems with surfaces of phospholipid bilayers patterned with TF have been used to demonstrate experimentally the threshold responses of initiation of coagulation to the size and shape of surfaces presenting TF. These systems have also been used to demonstrate experimentally that propagation of coagulation is regulated by the shear rate of blood flow in microcapillaries and microchannels. By understanding these and other aspects of the spatial dynamics that regulate blood coagulation, many new methods for treating clotting disorders, such as venous thromboembolism (VTE) and sepsis, could arise.

The solution structure and dynamics of the human coagulationfactor X (FX) have been investigated to understand the key structural elements in the zymogenic form that participates in the activation process. The model was constructed based on the 2.3-A-resolution x-ray crystallographic structure of active-site inhibited human FXa (PDB:1XKA). The missing gamma-carboxyglutamic acid (GLA) and part of epidermal growth factor 1 (EGF1) domains of the light chain were modeled based on the template of GLA-EGF1 domains of the tissue factor (TF)-bound FVIIa structure (PDB:1DAN). The activation peptide and other missing segments of FX were introduced using homology modeling. The full calcium-bound model of FX was subjected to 6.2 ns of molecular dynamics simulation in aqueous medium using the AMBER6.0 package. We observed significant reorientation of the serine-protease (SP) domain upon activation leading to a compact multi-domain structure. The solution structure of zymogen appears to be in a well-extended conformation with the distance between the calcium ions in the GLA domain and the catalytic residues estimated to be approximately 95 A in contrast to approximately 83 A in the activated form. The latter is in close agreement with fluorescence studies on FXa. The S1-specificity residues near the catalytic triad show significant differences between the zymogen and activated structures. PMID:11867437

Hemophilia is a serious rare disease that requires continuous management and treatment for which the medicine is costly at the annual average of 100 million KRW for an individual. The aim of this study was to investigate trends in the utilization of coagulationfactor (CF) used for hemophilia treatment using the National Health Insurance database from 2010 to 2013 in Korea and compare the utilization of CF with other countries. The consumption of CF per capita (IU) in Korea was not more than other countries with similar income to Korea. However, CF usage per patient IU was higher because the prevalence rate of hemophilia in Korea was lower than in other countries while the number of serious patients was much more. Therefore, it is difficult to say that the consumption of hemophilia medicine in Korea is higher than that in other countries. The consumption and cost of hemophilia medicine in Korea is likely to increase due to the increased utilization of expensive bypassing agents and the widespread use of prophylaxis for severe hemophilia. Even during the research period, it increased slightly and other countries show a similar trend. Thus, hemophilia patient management should accompany active monitoring on the health and cost outcomes of pharmaceutical treatment in the future. This study is expected to contribute to further insight into drug policies for other countries that face similar challenges with high price pharmaceuticals.

Hemostasia is a defense mechanism that protects vascular integrity, avoids blood loss, and maintains blood fluidity throughout the circulatory system. The biochemical processes leading to blood clot formation are complex, and alterations can appear at any point within the chain of events. While a range of alterations can affect the coagulationfactors, some are more common than others in the general population, including congenital (hemophilia A and B, Von Willebrand's disease) and acquired disorders (anticoagulant drugs). Such diseases require special consideration in the context of dental treatment, and therefore must be known to dental professionals. Interconsultation with the hematologist will provide orientation on the characteristics of the disease and on the best approach to treatment, including the need for replacement therapy, the application of local hemostatic measures, the modification of anticoagulant therapy, etc. In any case, the most important concern is the prevention of bleeding complications by compiling a detailed clinical history, with adequate planning of treatment, and taking special care to avoid soft tissue damage during the dental treatment of such patients. The dental surgeon must enhance awareness among patients and their relatives of the importance of correct oral hygiene, which will help avoid the need for invasive dental treatments and will reduce the number of visits to the dentist.

Gravity is a potential factor of aggregative stability and/or coagulation for any heterogeneous system having a density contrast between the dispersed phase and its dispersion medium. However, gravity becomes comparable to other stability factors only when the particle size becomes large enough. Since the particle size may grow in time due to various other instabilities, even nano-systems may eventually become susceptible to gravity. There have been many attempts in the last century to incorporate gravity in the overall theory of aggregative stability, but the relevant papers are scattered over a wide variety of journals, some of which are very obscure. Reviews on this subject in modern handbooks are scarce and inadequate. No review describes the role of gravity at all three levels introduced by DLVO theory for characterizing aggregative stability, namely: particle pair interaction, collision frequency and population balance equation. Furthermore, the modern tendency towards numerical solutions overshadows existing analytical solutions. We present a consistent review at each DLVO level. First we describe the role of gravity in particle pair interactions, including both available analytical solutions as well as numerical stability diagrams. Next we discuss a number of works on collision frequency, including works for both charged and non-charged particles. Finally, we present analytical solutions of the population balance equation that takes gravity into account and then compare these analytical solutions with numerical solutions. In addition to the traditional aggregate model we also discuss work on a fractal model and its relevance to gravity controlled stability. Finally, we discuss many experimental works and their relationship to particular theoretical predictions.

Presurgical coagulation diagnosis should--apart from coagulation monitoring in the laboratory based on a stepwise diagnosis for detection of coagulations disorders, starting with global tests (NT/APTT) followed by appropriate specific investigation in case of pathological findings--consist of an adequate hemostaseological anamnesis and physical checkup of the patient. This would allow detection of important signs of hemostaseological impairment during the pre-analytical phase already and permit subsequent initiation of more specific coagulation tests. The casuistics of a patient with factor XI-deficiency ("Minor Form"), a condition which is extremely infrequent in our country, demonstrates the coagulation diagnostic procedure which led to detection of his inherited factor XI-deficiency. In addition the pre-, peri- and postsurgical therapeutical management of this particular patient using an antifibrinolytic drug (tranexamic acid) is presented.

The coagulation/fibrinolysis system is essential for wound healing after vascular injury. According to the standard paradigm, the synthesis of most coagulationfactors is restricted to liver, platelets and endothelium. We challenged this interpretation by measuring coagulationfactors in nine human primary cell types. FX mRNA was expressed by fibroblasts, visceral preadipocytes/adipocytes and hepatocytes, but not in macrophages or other cells. All cells expressed FVIII except endothelial cells. Fibroblasts, endothelial cells and macrophages produced thrombomodulin but not FV. Interestingly, vascular-related cells (platelets/monocytes) that expressed FV did not express FX and vice versa. Monocytes expressed FV, FVIII and FXIIIA, which are positive regulators of clot formation, but these cells also contained thrombomodulin, a negative regulator of coagulation. Our data show that the expression of coagulationfactors is much more complex than previously thought, and we speculate that this intricate regulation of coagulationfactor expression is necessary for correct fine-tuning of fibrinogenesis versus fibrinolysis. PMID:23145311

OBJECTIVE The aim of this study was to identify the participation of the coagulation system in the differential diagnosis of pleural effusions. INTRODUCTION Imbalance between immunologic and metabolic factors triggers a sequence of events resulting in pleural reactions and accumulation of fluid. The coagulation system, which is fundamental for the maintenance of homeostasis, contributes to the inflammatory process responsible for pleural effusions, and participates in cellular proliferation and migration as well as in the synthesis of inflammatory mediators. METHODS We evaluated the laboratory profile of coagulation and fibrinolysis in 54 pleural fluids (15 transudates and 39 exudates). RESULTS The coagulation system acts according to the pathophysiologic mechanisms involved in the development of pleural effusions. In inflammatory effusions (exudates), there is activation of coagulation with increased levels of fragment 1+2 and thrombin-antithrombin complex in addition to reduction of fibrinogen levels due to fibrinolysis and fibrin tissue incorporation. As a consequence, there is activation of the fibrinolytic system with increased levels of fibrin degradation products, including the D-dimer. These changes are not sufficient for differentiation of different subgroups of exudates. In transudates, these events were observed to a lesser degree. CONCLUSION The coagulation system plays an important role in the development of pleural diseases. Coagulation tests show differences between transudates and exudates but not among exudate subgroups. Understanding the physiopathological mechanisms of pleural disorders may help to define new diagnostic and therapeutic approaches. PMID:19759883

Patients with severe infections almost invariably exhibit evidence of activation of the coagulation system. The lungs are amongst the most frequently affected organs during severe infection and sepsis. The abundant presence of intravascular and extravascular fibrin appears to be a specific hallmark of acute lung injury after sepsis. Tissue factor (TF) is regarded to be the primary initiator of coagulation in severe infection. Effective blockade of the TF pathway, either by recombinant TF pathway inhibitor or by anti-TF antibodies in experimental sepsis, attenuates lung injury and partially prevents pulmonary dysfunction. In addition, inhibition of the activity of TF prevents local activation of coagulation in models of pneumonia. The TF pathway can influence inflammatory signaling by activation of protease activated receptor-1 and -2. This review presents the most recent data on the crosstalk between TF-mediated coagulation and inflammation, with a specific emphasis on these processes in the lung.

The automated laboratory analyzer COAGTRON-350 (Trinity Biotech) is used for routine and specific coagulation testing for the detection of fibrin formation utilizing either mechanical principles (ball method) or photo-optical principles, chromogenic kinetic enzyme analysis, and immune-turbidimetric detection systems in one benchtop unit. In this study, we demonstrated and established a parameter for the measurement of factor XIII (FXIII) activity using Berichrom FXIII reagent and the COAGTRON-350 analyzer. The usual protocol used for this reagent, based on the handling method, was slightly modified for this device. The analysis showed that fundamental study for the measurement of FXIII activity under our condition setting was favorable in terms of reproducibility, linearity, and correlation with another assays. Since FXIII is the key enzyme that plays important roles in hemostasis by stabilizing fibrin formation, the measurement of FXIII is essential for the diagnosis of bleeding disorders. Therefore, FXIII activity assessment as well as a routine coagulation testing can be conducted simultaneously with one instrument, which is useful in coagulopathy assessment.

Allometric scaling is a useful tool in early drug development and can be used for the prediction of human pharmacokinetic (PK) parameters from animal PK parameters. The main objective of this work was to predict concentration-time profiles of coagulationfactors in humans in a multi-compartment system using animal PK parameters. The prediction of concentration-time profiles in humans in a multi-compartment system was based on the predicted values of clearance and volumes of distribution (V(c), V(ss) and V(β)) from animals. Five coagulationfactors from the literature were chosen that were described by two-compartment model in both humans and animals. Clearance and volumes of distribution from animals were allometrically scaled to humans and then were used to predict concentration-time profiles in humans. The predicted concentration-time profile for a given coagulationfactor was accurate for most of the time points. Percent prediction error range varied across coagulationfactors. The prediction error >50% was observed either at 1 or a maximum of two time points for a given drug. The study indicated that the allometric scaling can be useful in the prediction of concentration-time profiles of coagulationfactors in humans from animals and may be helpful in designing a first-in-human study.

The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage–specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulationfactors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection. PMID:26341257

The activated partial thromboplastin time (aPTT) is a widely used coagulation screening test in routine laboratories. The aPTT level in the control population varies and is reflected by the reference interval. However, there have been no studies to investigate the coagulation status determining the variability of the aPTT. The aim of this study was to investigate the coagulationfactor activities underlying the variability of aPTT in the population. The study participants were reference individuals with prothrombin time and aPTT within reference intervals. The aPTT was determined using STA-PTT Automate (Diagnostica Stago, Asnieres, France; local reference interval, 29.1-41.9 s). Those with aPTT within the marginal ranges of reference interval were selected for factor assays. We defined the lower marginal group as the lowest 10 percentile of reference interval (29.1-30.9 s) and the upper marginal group as the highest 10 percentile (38.0-41.9 s). Activities of factor II, V, VIII, IX, X, XI, and XII were determined in both groups. The lower marginal and upper marginal groups consisted of 220 and 209 individuals, respectively. All coagulationfactors were significantly higher in the lower marginal than in the upper marginal group (P = 0.0127 for factor II and P < 0.0001 for the others). Multiple logistic regression analyses revealed factor XII and VIII were two strongest contributors determining the aPTT level, whereas factor XI was not significantly different between the groups (P = 0.095). This study firstly demonstrated significantly different coagulationfactor activities underlying the variability of aPTT in reference individuals. The results suggested the possibility of disease association or phenotypic contribution of variable coagulation activities in the population.

It has been suggested that the main physiological trigger of coagulation, tissue factor, possesses limited procoagulant activity and occurs in an inactive or so-called encrypted state. For the conversion of encrypted into decrypted tissue factor with sufficient procoagulant activity, four distinct models have been proposed: 1; dimer formation, 2; lipid rafts, 3; disulfide bonds, and 4; phosphatidylserine exposure. Pro and cons can be given for each of these mechanisms of tissue factor encryption/decryption, however, it seems most likely that two or more mechanisms act together in activating the procoagulant activity. The exposure of phosphatidylserine in the outer layer of cell membranes supports coagulation through enhanced formation of the tenase (factors IXa, VIIIa and X) and prothrombinase (factors Xa, Va and prothrombin) complexes. The proposed role for phosphatidylserine in decryption of tissue factor could contribute to the correct orientation of the tissue factor - factor VII complex. Overall, the contribution of both tissue factor and phosphatidylserine to coagulation seems distinct with tissue factor being the physiological activator and phosphatidylserine the driving force of propagation of coagulation.

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins of Staphylococcus aureus. We have shown that SSL10 binds to vitamin K-dependent coagulationfactors and inhibits blood coagulation induced by recalcification of citrated plasma. SSL10 was revealed to bind to coagulationfactors via their γ-carboxyglutamic acid (Gla) domain. In this study we attempted to identify the responsible sequence of SSL10 for the interaction with coagulationfactors. We prepared a series of domain swap mutants between SSL10 and its paralog SSL7 that does not interact with coagulationfactors, and examined their binding activity to immobilized prothrombin using ELISA-like binding assay. The domain swap mutants that contained SSL10β1-β3 ((23)MEMKN ISALK HGKNN LRFKF RGIKI QVL(60)) bound to immobilized prothrombin, and mutants that contained SSL10β10-β12 ((174)SFYNL DLRSK LKFKY MGEVI ESKQI KDIEV NLK(207)) also retained the binding activity. On the other hand, mutants that lacked these two regions did not bind to prothrombin. These sequences, each alone, bound to prothrombin as 33 amino acid length polypeptides. These results suggest that SSL10 has two responsible sequences for the binding to prothrombin. These prothrombin-binding peptides would contribute to the development of new anticoagulants.

Haemostasis prevents blood loss following vascular injury. It depends on the unique concert of events involving platelets and specific blood proteins, known as coagulationfactors. The clotting system requires precise regulation and coordinated reactions to maintain the integrity of the vasculature. Clotting insufficiency mostly occurs due to genetically inherited coagulationfactor deficiencies such as hemophilia. A relevant literature search of PubMed was performed using the keywords coagulationfactors, Nonsense-mediated mRNA decay and premature translation termination codons. Search limitations included English language and human-based studies. Mutations that cause premature translation termination codons probably account for one-third of genetically inherited diseases. Transcripts bearing aberrant termination codons are selectively identified and eliminated by an evolutionarily conserved posttranscriptional pathway known as nonsense-mediated mRNA decay (NMD). There are many pieces of evidence of decay among coagulationfactor genes. However, the hemophilia gene (F8) does not seem to be subjected to NMD. Since the F8 gene is located on the X-chromosome, a connection between X-linked traits and mRNA decay could be assumed. Considering that not all genes go through decay, this review focuses on the basics of the mechanism in coagulation genes. It is interesting to determine whether this translation-coupled surveillance system represents a general rule for the genes encoding components of the same physiological cascade.

Objective(s): Haemostasis prevents blood loss following vascular injury. It depends on the unique concert of events involving platelets and specific blood proteins, known as coagulationfactors. The clotting system requires precise regulation and coordinated reactions to maintain the integrity of the vasculature. Clotting insufficiency mostly occurs due to genetically inherited coagulationfactor deficiencies such as hemophilia. Materials and Methods: A relevant literature search of PubMed was performed using the keywords coagulationfactors, Nonsense-mediated mRNA decay and premature translation termination codons. Search limitations included English language and human-based studies. Results: Mutations that cause premature translation termination codons probably account for one-third of genetically inherited diseases. Transcripts bearing aberrant termination codons are selectively identified and eliminated by an evolutionarily conserved posttranscriptional pathway known as nonsense-mediated mRNA decay (NMD). There are many pieces of evidence of decay among coagulationfactor genes. However, the hemophilia gene (F8) does not seem to be subjected to NMD. Since the F8 gene is located on the X-chromosome, a connection between X-linked traits and mRNA decay could be assumed. Conclusion: Considering that not all genes go through decay, this review focuses on the basics of the mechanism in coagulation genes. It is interesting to determine whether this translation-coupled surveillance system represents a general rule for the genes encoding components of the same physiological cascade. PMID:27279976

When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikrein-kinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates. PMID:18725990

Coagulationfactor XI is present in blood plasma as the zymogen, like other serine proteases of hemostatic system, but as the only coagulationfactor forms 140-160kDa homodimers. Its activation is induced by thrombin, and a positive feedback increases the generation of the extra thrombin. Experimental and clinical observations confirm protective roles of factor XI deficiencies in certain types of thromboembolic disorders. Thromboembolism still causes serious problems for modern civilization. Diseases associated with the blood coagulation system are often associated with inflammation and oxidative stress. Peroxynitrite is produced from nitric oxide and superoxide in inflammatory diseases. The aim of the current study is to evaluate effects of nitrative stress triggered by peroxynitrite on coagulationfactor XI in human plasma employing biochemical and bioinformatic methods. The amidolytic assay shows increase in factor XI activity triggered by peroxynitrite. Peroxynitrite interferes factor XI by nitration and fragmentation, which is demonstrated by immunoprecipitation followed by western blotting. Nitrated factor XI is even present in control blood plasma. The results suggest possible modifications of factor XI on the molecular level. Computer simulations show tyrosine residues as targets of peroxynitrite action. The modifications induced by peroxynitrite in factor XI might be important in thrombotic disorders.

To investigate the risk factors for early disseminated intravascular coagulation (DIC) in neonates with sepsis. A retrospective clinical study was performed on 100 neonates with a confirmed diagnosis of sepsis between 2012 and 2013. The children were classified into normal coagulation group, non-overt DIC group (early DIC group), and overt DIC group (late DIC group) based on the ISTH overt DIC scoring system. The clinical manifestations and risk factors were analyzed statistically. Early DIC occurred in 44 (44%) cases in the 100 neonates with sepsis. The incidence of sclerema showed significant differences between the three groups (P<0.05). Asphyxia, bleeding, and Gram-negative bacterial infection were independent risk factors for early DIC. Coagulation function should be actively monitored and early intervention measures should be taken for neonates with asphyxia, bleeding, and Gram-negative bacterial infection to prevent early DIC from progressing to late DIC.

We studied the blood coagulation system of 14 patients with metastatic malignancies before and after they had undergone major surgery. In addition to measuring a battery of coagulationfactors, we assessed the function of the system with assays of whole blood thrombin generation. With the exceptions of factor VIII (fVIII), which increased, and fibrinogen and fIX, which did not change, the activities of all the pro- and anticoagulant proteins were significantly lower postoperatively. However, the thrombin generating capacity of the system was relatively preserved. Although the integral of thrombin activity over time was lower after surgery, the mean peak thrombin concentration was unchanged and the time to clot formation was shortened. Similar changes could be reproduced by lowering the concentrations of pro- and anticoagulant factors together in control blood samples. Therefore, simultaneous reductions in pro- and anticoagulant proteins postoperatively worked to maintain the functional integrity of the blood coagulation system. 2007 Wiley-Liss, Inc

Metastatic cancer is associated with a hypercoagulable state, and pathological venous thromboembolic disease is a significant source of morbidity and the second leading cause of death in patients with cancer. Here we aimed to develop a novel labeling strategy to detect and quantify procoagulant circulating tumor cells (CTCs) from patients with metastatic cancer. We hypothesize that the enumeration of procoagulant CTCs may be prognostic for the development of venous thrombosis in patients with cancer. Our approach is based on the observation that cancer cells are capable of initiating and facilitating cell-mediated coagulation in vitro, whereby activated coagulationfactor complexes assemble upon cancer cell membrane surfaces. Binding of fluorescently labeled, active site-inhibited coagulationfactors VIIa, Xa, and IIa to the metastatic breast cancer cell line, MDA-MB-231, non-metastatic colorectal cell line, SW480, or metastatic colorectal cell line, SW620, was characterized in a purified system, in anticoagulated blood and plasma, and in plasma under conditions of coagulation. We conclude that a CTC labeling strategy that utilizes coagulationfactor-based fluorescent probes may provide a functional assessment of the procoagulant potential of CTCs, and that this strategy is amenable to current CTC detection platforms. PMID:22973554

The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

The autosomal dominantly inherited east Texas bleeding disorder is linked to an A2440G variant in exon 13 of the F5 gene. Affected individuals have normal levels of coagulationfactor V (FV) activity, but demonstrate inhibition of global coagulation tests. We demonstrated that the A2440G mutation causes upregulation of an alternatively spliced F5 transcript that results in an in-frame deletion of 702 amino acids of the large activation fragment, the B domain. The approximately 250-kDa FV isoform (FV-short), which can be fully activated by thrombin, is present in all A2440G carriers' plasma (n = 16). FV-short inhibits coagulation through an indirect mechanism by forming a complex with tissue factor pathway inhibitor-α (TFPIα), resulting in an approximately 10-fold increase in plasma TFPIα, suggesting that the TFPIα:FV-short complexes are retained in circulation. The TFPIα:FV-short complexes efficiently inhibit thrombin generation of both intrinsic and extrinsic coagulation pathways. These data demonstrate that the east Texas bleeding disorder-associated F5(A2440G) leads to the formation of the TFPIα:FV-short complex, which inhibits activation and propagation of coagulation.

The autosomal dominantly inherited east Texas bleeding disorder is linked to an A2440G variant in exon 13 of the F5 gene. Affected individuals have normal levels of coagulationfactor V (FV) activity, but demonstrate inhibition of global coagulation tests. We demonstrated that the A2440G mutation causes upregulation of an alternatively spliced F5 transcript that results in an in-frame deletion of 702 amino acids of the large activation fragment, the B domain. The approximately 250-kDa FV isoform (FV-short), which can be fully activated by thrombin, is present in all A2440G carriers’ plasma (n = 16). FV-short inhibits coagulation through an indirect mechanism by forming a complex with tissue factor pathway inhibitor-α (TFPIα), resulting in an approximately 10-fold increase in plasma TFPIα, suggesting that the TFPIα:FV-short complexes are retained in circulation. The TFPIα:FV-short complexes efficiently inhibit thrombin generation of both intrinsic and extrinsic coagulation pathways. These data demonstrate that the east Texas bleeding disorder–associated F5A2440G leads to the formation of the TFPIα:FV-short complex, which inhibits activation and propagation of coagulation. PMID:23979162

Trauma patients present with a coagulopathy, termed early trauma-induced coagulopathy (ETIC), that is associated with increased mortality. This study investigated hemostatic changes responsible for ETIC. Case-control study of trauma patients with and without ETIC, defined as prolonged prothrombin time (PT), was performed from prospective cohort of consecutive trauma patients who presented to Level I trauma center. Univariate and multivariate analyses were performed. The case-control study group (n = 91) was 80% male, with mean age of 37 years, 17% penetrating trauma and 7% mortality rate. Patients with ETIC demonstrated decreased common and extrinsic pathway factor activities (factors V and VII) and decreased inhibition of the coagulation cascade (antithrombin and protein C activities) when compared with the matched control patients without ETIC. Both cohorts had evidence of increased thrombin and fibrin generation (prothrombin fragment 1.2 levels, thrombin-antithrombin complexes, and soluble fibrin monomer), increased fibrinolysis (d-dimer levels), and increased inhibition of fibrinolysis (plasminogen activator inhibitor-1 activity) above normal reference values. Patients with versus without ETIC had increased mortality and received increased amount of blood products. ETIC following injury is associated with decreased factor activities without significant differences in thrombin and fibrin generation, suggesting that despite these perturbations in the coagulation cascade, patients displayed a balanced hemostatic response to injury. The lower factor activities are likely secondary to increased hemodilution and coagulationfactor depletion. Thus, decreasing the amount of crystalloid infused in the early phases following trauma and administration of coagulationfactors may prevent the development.

A coagulationFactor V inhibitor developed in a man 75 yr of age in association with an anaplastic malignancy and drug treatment (including the aminoglycoside antibiotic, gentamicin). The patient did not bleed abnormally, despite both surgical challenge and plasma Factor V activity of less than 1%. The inhibited plasma had grossly prolonged prothrombin and activated partial thromboplastin times, but a normal thrombin time. Mixing studies indicated progressive coagulation inhibition with normal plasma, but not with Factor V-deficient plasma, and reversal of coagulation inhibition by the addition of bovine Factor V to the patient's plasma. 1 ml of patient plasma inhibited the Factor V activity of 90 ml of normal human plasma. The inhibitor was isolated by sequential affinity chromatography on protein A-Sepharose and Factor V-Sepharose. The IgG isolate markedly inhibits the activity of prothrombinase assembled from purified Factors Xa and Va, calcium ion, and phospholipid vesicles, and partially inhibits prothrombinase assembled from purified Factor Xa, calcium ion, and normal platelets. The Factor V of platelets, however, appears relatively inaccessible to the antibody, inasmuch as platelets isolated from whole blood supplemented for 8 h with the antibody functioned normally with respect to platelet Factor V-mediated prothrombinase function. The absence of obvious hemorrhagic difficulties in the patient, the total inhibition of plasma Factor V by the inhibitor, and the apparent inaccessibility of platelet Factor V to the inhibitor specifically implicate platelet Factor V in the maintenance of hemostasis. Images PMID:3944265

Combined deficiency of coagulationfactors is considered as an extremely rare bleeding disorder (RBD) inherited in an autosomal recessive pattern. This disorder is more likely to occur in regions with a high rate of consanguineous marriages or in restricted communities. Sistan and Baluchistan, a province in southeast of Iran with a high rate of consanguinity, is a clear model of such regions with a very high prevalence of recessively inherited disorders. The aim of this study was to report the frequency of combined factor deficiency in this province. This descriptive study was conducted on 358 patients with RBD. Demographic information and medical history of each patient were recorded, and the patients were examined by a physician. Routine screening tests were carried out for all patients, and further coagulation tests including coagulationfactor activity and antigen assays were subsequently performed for all suspected patients. Among 358 patients, four were found to be affected with combined factor (F)V and FVIII deficiency (F5F8D). In addition, one patient with combined deficiency of FVII-FXIII, one with combined FVII-FX and one with combined FVIII-FIX deficiency were identified. In Sistan and Baluchistan Province, coinheritance of recessively inherited disorders like combined coagulationfactor deficiencies was surprisingly higher than expected.

A plasmid containing human coagulationfactor VII (hFVII) complementary DNA regulated by a cytomegalovirus promoter was microinjected into fertilized eggs of zebrafish, African catfish, and tilapia. The active form of hFVll was detected in the fish embryos by various assays. This positive expression of human therapeutic protein in fish embryos demonstrates the possibility of exploitation of transgenic fish as bioreactors.

Hemostatic disorders presumptively play an important role in the pathophysiology of several important disease conditions in the Florida manatee ( Trichechus manatus latirostris). Prior to pursuing such clinical implications, it is essential to establish normal hemostatic profiles in clinically healthy animals. During annual health assessments of free-living manatees organized by the US Geological Survey, blood samples were collected from 12 healthy animals from the Atlantic coast and 28 from the Gulf of Mexico coast of Florida, with body lengths of 210-324 cm. The following analyses were performed on citrated plasma: prothrombin (PT), partial thromboplastin time (PTT), and concentrations of fibrinogen, D-dimers, and coagulationfactors VII, VIII, IX, X, XI, and XII. Compared to other mammalian species, manatees had short PT (9.2±1.5 s) and PTT (10.7±0.5 s), fibrinogen was 369±78.7 mg/dL, antithrombin III was 132±11%, and D-dimer was 142±122 ng/mL. Baseline concentrations for the listed coagulationfactors were established. When comparing coagulationfactors between locations, Atlantic coast manatees had significantly higher factors VIII, IX, and X than did Gulf Coast manatees. This finding may reflect differences in water salinity, diet, or genetics. There were no differences in coagulationfactors when among sexes and sizes. These baselines for hemostatic profiles and coagulationfactors in healthy free-living manatees lay the foundation for diagnosis and future research of hemostatic disorders and contribute to understanding their role in the pathophysiology of manatees affected by various diseases.

In 1991 it was demonstrated that, besides factor XII, thrombin is capable of activating factor XI in vitro. Thrombin-dependent activation of factor XI is an integral part of the revised theoretical model of coagulation in which coagulation is initiated by the extrinsic pathway and maintained by thrombin-induced activation of clotting factors V, VIII, and XI. In this review, special interest is given to the new role of factor XI in coagulation, with emphasise on data supporting the concept of thrombin-mediated factor XI activation in vivo. Furthermore, activation of factor XI in human disease, especially atherosclerotic disease, measured by newly developed immunologic assays, is discussed. The relation of factor XI to fibrinolysis through activation of the carboxypeptidase, thrombin-activatable fibrinolysis inhibitor (TAFI) by thrombin provides an explanation for the bleeding tendency observed in factor XI-deficient patients. The probable link with factor XI-mediated TAFI activation may have clinical and therapeutic consequences and deserves further study.

The ability to use plasma, isolated from units of whole blood and frozen within 24 h of phlebotomy, as a substitute for plasma frozen within 8 h of phlebotomy would have several advantages for blood centers. It should provide increased flexibility pertaining to the freezing of plasma for clinical use. We have conducted studies to assess the influence of an extended holding time for separated plasma, prior to freezing, on the retention of coagulationfactor activity. Freshly harvested plasma from each of 10 units of CPD-whole blood was divided into four equal aliquots. These aliquots were held in plastic packs at 1-6 degrees C for a total of 0, 8, 15 and 24 h. Subsequently, the plasma aliquots were frozen rapidly and stored at -20 degrees C for 4 months. The thawed plasma was tested for coagulantfactors V and IX, factor VIII coagulant activity (factor VIII:C), von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor of von Willebrand factor. The levels of factor V, factor vWF:Ag, factor IX and ristocetin cofactor were not influenced by holding the plasma for up to 24 h prior to freezing. Factor VIII:C activity was reduced with extended holding at 1-6 degrees C; the percentage at time zero activity was 75.9+/-2.4% for samples frozen immediately after a 24-hour period. The data indicate that coagulationfactor properties of harvested plasma are retained except for factor VIII for at least 24 h prior to freezing.

We have independently isolated and characterized cDNA and genomic clones for the human coagulationfactor IX. Sequence analysis in both cases indicates that threonine is encoded by the triplet ACT as the third residue of the activation peptide. This is in agreement with some earlier reports but in disagreement with others that show the alanine triplet GCT at this position. The discrepancy can thus be accounted for by natural variation of a single nucleotide in the normal population. Amino acid sequence analyses of activated factor IX from plasma samples of four individuals yielded two cases of alanine and two cases of threonine at the third position of the activation peptide. In factor IX from pooled plasma and in factor IX from a heterozygous individual, however, both alanine and threonine were found. Taken together, the findings show that a prevalent nondeleterious dimorphism exists in the activation peptide of human coagulationfactor IX. PMID:3857619

To evaluate the comparability of measurement results for coagulationfactor VIII (FVIII)and factor IX (FIX) activity and the commutability of reference materials on different measurement systems. The study was performed according to CLSI guideline EP30 and China health standard WS/T 356-2011. Clinical samples with different levels of FVIII and FIX which covered over the clinical analytical range, five lots of homemade reference materials (F20140601-F20140605) and a coagulation reference material (SSCLOT4) provided by NIBSC were detected for FVIII and FIX activity on three popular measurement systems in China, which including Stago STA-R Evolution, IL ACL TOP700 and Sysmex CA7000 automatic coagulation analyzers using supplementary reagents. The results between measurement systems were analyzed pairwise. To evaluate the comparability, the linear regression and the biases between the results of clinical samples from two measurement systems were calculated. The comparability was evaluated by the regression coefficient and the biases inside the acceptable range. After eliminated outliers from the results, linear regressions were run again and the 95% confidence intervals were calculated. The commutability of the homemade reference materials and NIBSC reference material were evaluated by comparing the results with the limits of the intervals. The ranges of FVIII and FIX level of clinical samples were 0.5%-218.0% and 1.6%-156.5%, which covered the sample levels in routine work and fit the requirements for commutability evaluation. The square of correlation coefficients (R²) of measurement results of clinical samples for FVIII and FIX activity assays were 0.89-0.94 and 0.81-0.93. The proportions of outliers were all less than 10%. The comparability of measurement results of FVIII and FIX in different measurement systems was acceptable.According to the acceptable criteria for bias, the measurement results of 42, 41 and 45 clinical samples for FVIII and 44, 42 and 41

The risk of venous thromboembolism is increased in patients with lymphoma and leukemia; however, little is known about the potential underlying hereditary or acquired thrombophilia. We prospectively analyzed procoagulant markers and gene mutations in patients with lymphoma (n = 35) and leukemia (n = 10) at diagnosis and over the course of treatment. Global coagulation tests were normal in all patients, as were antithrombin and protein S. Activated protein C resistance caused by the factor V Leiden mutation was found in four patients, one patient had the G20210A mutation of the prothrombin gene, and one patient had protein C deficiency. The most striking findings were sustained very high levels of factor VIII (>150 %) in 30 patients (68 %), which correlated with high von Willebrand factor. An acute phase response in these patients was ruled out by absence of fever and normal IL-6 and -α. Elevated factor VIII is an independent thrombophilic risk factor and may play an etiologic role in thromboembolic complications in patients with malignant lymphoma. Since high von Willebrand factor is most likely caused by endothelial cell injury, an additional, unknown pathophysiological association with malignant lymphoma and acute leukemia is possible.

Binding of coagulationfactors to phosphatidylserine (PS)-exposing procoagulant-activated platelets followed by formation of the membrane-dependent enzyme complexes is critical for blood coagulation. Procoagulant platelets formed upon strong platelet stimulation, usually with thrombin plus collagen, are large "balloons" with a small (∼1 μm radius) "cap"-like convex region that is enriched with adhesive proteins. Spatial distribution of blood coagulationfactors on the surface of procoagulant platelets was investigated using confocal microscopy. All of them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin V were distributed nonhomogeneously: they were primarily localized in the "cap," where their mean concentration was by at least an order of magnitude, higher than on the "balloon." Assembly of intrinsic tenase on liposomes with various PS densities while keeping the PS content constant demonstrated that such enrichment can accelerate this reaction by 2 orders of magnitude. The mechanisms of such acceleration were investigated using a 3-dimensional computer simulation model of intrinsic tenase based on these data. Transmission electron microscopy and focal ion beam-scanning electron microscopy with Annexin V immunogold-labeling revealed a complex organization of the "caps." In platelet thrombi formed in whole blood on collagen under arterial shear conditions, ubiquitous "caps" with increased Annexin V, FX, and FXa binding were observed, indicating relevance of this mechanism for surface-attached platelets under physiological flow. These results reveal an essential heterogeneity in the surface distribution of major coagulationfactors on the surface of procoagulant platelets and suggest its importance in promoting membrane-dependent coagulation reactions.

Some of the major issues related to the development and control of antibodies that occur during treatment of haemophilia with replacement factors (Factor VIII and Factor IX) are reviewed. Information on analytical issues, immunogenicity, and immune tolerance may be applicable to the study of other therapeutic proteins. Conversely, new information obtained from evaluation of other therapeutic protein products may address issues that remain unresolved for Factor VIII and FIX replacement therapy.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 1, the World Health Organisation (WHO) 3rd International Standard, Human (IS, 96/854) and the FDA Standard for human blood coagulationFactor IX concentrate have been available since 1996, following their establishment by a common collaborative study. Due to dwindling stocks of all three standards, a new WHO-EDQM-FDA tri-partite collaborative study was launched to establish replacement batches. Thirty laboratories from fourteen countries took part in the collaborative study to assign potency values to candidate preparations. Three candidates, one of recombinant and two of human plasma-derived origins, were assayed against the 3rd IS for Blood CoagulationFactor IX, Concentrate, Human (96/854). The 3rd IS for Blood CoagulationFactors II, VII, IX and X, Plasma, Human (99/826) was also included to evaluate the relationship between the factor IX plasma and concentrate unitage. Thirty-two sets of clotting assay results and two sets of chromogenic assay data were analysed. There was a significant difference in potency estimates by these two methods for the recombinant candidate (sample B) and the plasma IS (sample P). Similar potency values were obtained for the plasma derived products (monoclonal antibody- and chromatography-purified factor IX, samples C and D) by clotting and chromogenic assays. For the clotting assays, intra-laboratory variability (GCV) was found to range from 0.5 - 21.7%, with the GCV for the majority of laboratories being less than 10%. Good inter-laboratory agreement, with the majority of the GCV being less than 10% (GCV range = 4.7 - 10.6 %) was also obtained. The mean potency values estimated by the clotting assay using plasma as pre-diluent (as directed by the Ph. Eur. general chapter method) did not differ from values obtained using buffer. Taking into account the preliminary stability data, the intra- and inter-laboratory variability, and the differences

Non-activated coagulationfactor IX was purified approx. 10,000-fold from human plasma. The final product was electrophoretically homogeneous and comprised a tingle polypeptide chain with a molecular weight of about 70,000 and a pI of 4.3-4.45. The N-terminal amino acid was glycine. The amino acid and the carbohydrate contents were analysed and a monospecific antiserum to the factor was raised in rabbits. Images PLATE 1 PLATE 2 PMID:1171684

Purified human Factor X (apparent mol.wt. 72000), which consists of two polypeptide chains (mol.wt. 55000 and 19000), was activated by both Russell's-viper venom and the purified physiological activators (Factor VII/tissue factor and Factor IXa/Factor VIII). They all convert Factor X to catalytically active Factor Xa (mol.wt. 54000) by cleaving the heavy chain at a site on the N-terminal region. In the presence of Ca2+ and phospholipid, the Factor Xa formed catalyses (a) the cleavage of a small peptide (mol.wt. 4000) from the C-terminal region of the heavy chain of Factor Xa, resulting in a second active form (mol.wt. 50000), and (b) the cleavage of a peptide containing the active-site serine residue (mol.wt. 13000) from the C-terminal region of the heavy chain of Factor X, resulting in an inactivatable component (mol.wt. 59000). A nomenclature for the various products is proposed. PMID:6770848

Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. Expression of coagulationfactor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/- SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/-15% to 188+/-27% and 100+/-8.8% to 176.3+/-17.3% respectively, p<0.001) at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress.

Background Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. Methodology/Principal Findings Expression of coagulationfactor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/− SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/−15% to 188+/−27% and 100+/−8.8% to 176.3+/−17.3% respectively, p<0.001) at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. Conclusions/Significance Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress. PMID:22848420

We present herein a case of a young female with congenital combined coagulationfactor VII (FVII) and factor II (FII) deficiencies. She was completely asymptomatic and found to have a prolonged prothrombin time during a routine preoperative evaluation. Low levels of plasma FVII and FII in the absence of an inhibitor confirmed the diagnosis in our patient. Congenital combined FVII and FIX deficiency as well as combined FVII and FX deficiency have been previously reported. The congenital combined deficiency of FVII and FII in our patient is exceptional and represents the first such instance in the English literature. Furthermore, we hypothesize that she had not shown any bleeding manifestations because of possible compensation for the missing factors II and VII by enhanced activity of some intrinsic coagulation pathway components or depression of fibrinolysis.

Recent studies have shown that acute traumatic coagulopathy is associated with hypoperfusion, increased plasma levels of soluble thrombomodulin, and decreased levels of protein C but with no change in factor VII activity. These findings led to the hypothesis that acute traumatic coagulopathy is primarily due to systemic anticoagulation, by activated protein C, rather than decreases in serine protease activity. This study was designed to examine the effect of hypoperfusion secondary to traumatic injury on the activity of coagulationfactors. Post hoc analysis of prospectively collected data on severely injured adult trauma patients presenting to a single trauma center within 120 minutes of injury. Venous blood was analyzed for activity of factors II, V, VII, VIII, IX, X, and XI. Base deficit from arterial blood samples was used as a marker of hypoperfusion. Seventy-one patients were identified. The activity of factors II, V, VII, IX, X, and XI correlated negatively with base deficit, and after stratification into three groups, based on the severity of hypoperfusion, a statistically significant dose-related reduction in the activity of factors II, VII, IX, X, and XI was observed. Hypoperfusion is also associated with marked reductions in factor V activity levels, but these appear to be relatively independent of the degree of hypoperfusion. The activity of factor VIII did not correlate with base deficit. Hypoperfusion in trauma patients is associated with a moderate, dose-dependent reduction in the activity of coagulationfactors II, VII, IX, X, and XI, and a more marked reduction in factor V activity, which is relatively independent of the severity of shock. These findings suggest that the mechanisms underlying decreased factor V activity--which could be due to activated protein C mediated cleavage, thus providing a possible link between the proposed thrombomodulin/thrombin-APC pathway and the serine proteases of the coagulation cascade--and the reductions in factors

Hereditary angioedema (HAE) is a rare disorder characterized by recurrent, acute, and painful episodes of swelling involving multiple tissues. Deficiency or malfunction of the serine protease inhibitor C1 esterase inhibitor (C1-INH) results in HAE types 1 and 2, respectively, whereas mutations in coagulationfactor 12 (f12) have been associated with HAE type 3. C1-INH is the primary inhibitor of multiple plasma cascade pathways known to be altered in HAE patients, including the complement, fibrinolytic, coagulation, and kinin-kallikrein pathways. We have selectively inhibited several components of both the kinin-kallikrein system and the coagulation cascades with potent and selective antisense oligonucleotides (ASOs) to investigate their relative contributions to vascular permeability. We have also developed ASO inhibitors of C1-INH and characterized their effects on vascular permeability in mice as an inducible model of HAE. Our studies demonstrate that ASO-mediated reduction in C1-INH plasma levels results in increased vascular permeability and that inhibition of proteases of the kinin-kallikrein system, either f12 or prekallikrein (PKK) reverse the effects of C1-INH depletion with similar effects on both basal and angiotensin converting enzyme (ACE) inhibitor-induced permeability. In contrast, inhibition of coagulationfactors 11 (f11) or 7 (f7) had no effect. These results suggest that the vascular defects observed in C1-INH deficiency are dependent on the kinin-kallikrein system proteases f12 and PKK, and not mediated through the coagulation pathways. In addition, our results highlight a novel therapeutic modality that can potentially be employed prophylactically to prevent attacks in HAE patients.

The coagulation cascade plays a critical role in the development of cardiovascular disease (CVD). Elevated plasma prothrombin fragment 1 + 2 (F1 + 2) and factor VIII antigen (FVIII:Ag) levels have been associated with a hypercoagulable state, enhancing the risk for vascular thrombotic events. Aerobic training is known to reduce CVD risk, and an improved coagulation profile may contribute to this reduction. To analyze the effect of 6 months of standardized aerobic exercise training on resting F1 + 2 and FVIII:Ag levels in men and postmenopausal women aged 50-75 while accounting for several possibly confounding factors. Sedentary men (N=16) and women (N=31) underwent supervised aerobic training 3 d x wk(-1) for 6 months while maintaining the American Heart Association step 1 diet. Baseline and final testing included measurement of F1 + 2, FVIII:Ag, plasma lipoprotein-lipid levels, body composition, and VO2max. When adjusted for baseline values and changes in diastolic blood pressure with training, F1 + 2 was found to decrease significantly with exercise training from 1.493 +/- 0.058 to 1.422 +/- 0.059 nM (P=0.014). FVIII:Ag levels were found to increase significantly with training when adjusted for baseline values, from 152.5 +/- 6.7% of standard at baseline to 156.0 +/- 6.1% of standard at final testing (P=0.005). Training-induced changes in coagulation markers were independent of changes in blood lipids, aerobic capacity, and body composition. : These results indicate that endurance training has a significant impact on the coagulation cascade, reducing coagulation activity in the common pathway and thrombin formation at rest while increasing the activation potential of the intrinsic pathway.

Background Trauma patients present with a coagulopathy, termed early trauma induced coagulopathy (ETIC), which is associated with increased mortality. This study investigated hemostatic changes responsible for ETIC. Methods Case-control study of trauma patients with and without ETIC, defined as prolonged prothrombin time (PT), was performed from prospective cohort of consecutive trauma patients who presented to level I trauma center. Univariate and multivariate analyses were performed. Results The case control study group (n=91) was 80% male, mean age of 37 years, 17% penetrating trauma and 7% mortality rate. ETIC patients demonstrated decreased common and extrinsic pathway factor activities (factors V and VII) and decreased inhibition of the coagulation cascade (antithrombin and protein C activities) as compared to the matched control non-ETIC patients. Both cohorts had evidence of increased thrombin and fibrin generation (prothrombin fragment 1.2 levels, thrombin-antithrombin complexes, soluble fibrin monomer,), increased fibrinolysis (D-dimer levels) and increased inhibition of fibrinolysis (plasminiogen activator inhibitor-1 activity) above normal reference values. ETIC versus non-ETIC patients had increased mortality and received increased amount of blood products. Conclusion ETIC following injury is associated with decreased factor activities without significant differences in thrombin and fibrin generation suggesting that despite these perturbations in the coagulation cascade patients displayed a balanced hemostatic response to injury. The lower factor activities are likely secondary to increased hemodilution and coagulationfactor depletion. Thus, decreasing the amount of crystalloid infused in the early phases following trauma and administration of coagulationfactors may prevent the development. PMID:21460741

Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR) or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin) as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT) imaging of gene expression to determine whether local virus administration (direct injection in the kidney) could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting. PMID:23638001

I L I M NaCI, 2) 2 L distilled water, and 3) 2 L QAE buffer using moderate vacuum. The caramel - colored supernatant was decanted into a 20 gallon...a thrombin substrate, which when cleaved provides a color change, was used with results as shown in Figures 22 and 23. The low levels of thrombin...indicated an increase, with time, of factor V/Va antigen in culture supernatants, and a constant level of antigen per cell from solubilized cells. Not all

A simple microtechnique for carrying out partial thromboplastin time with kaolin tests with 2 microliter or less of test plasma is described. For single stage factor assays, less than 1 microliter of test solution may be used. Reagents and test plasma are loaded in sequence into a 10 microliter, long needle syringe and introduced into a micro test-tube immobilized in a water bath. The end-point is taken as a positive clearing of kaolin turbidity from the mixture while stirring. Correlation with normal techniques has been excellent.

Blood coagulation is triggered not only by surface tissue factor (TF) density but also by surface TF distribution. We investigated recognition of surface TF distribution patterns during blood coagulation and identified the underlying molecular mechanisms. For these investigations, we employed 1), an in vitro reaction-diffusion experimental model of coagulation; and 2), numerical simulations using a mathematical model of coagulation in a three-dimensional space. When TF was uniformly immobilized over the activating surface, the clotting initiation time in normal plasma increased from 4 min to >120 min, with a decrease in TF density from 100 to 0.7 pmol/m2. In contrast, surface-immobilized fibroblasts initiated clotting within 3–7 min, independently of fibroblast quantity and despite a change in average surface TF density from 0.5 to 130 pmol/m2. Experiments using factor V-, VII-, and VIII-deficient plasma and computer simulations demonstrated that different responses to these two TF distributions are caused by two positive feedback loops in the blood coagulation network: activation of the TF–VII complex by factor Xa, and activation of factor V by thrombin. This finding suggests a new role for these reactions: to supply sensitivity to local TF density during blood coagulation. PMID:22004734

Thrombosis complicating the rupture of an atherosclerotic plaque can lead to arterial occlusion. Tissue factor, a membrane-bound glycoprotein, is expressed to a greater extent in atherosclerotic plaques and may be a key mediator of microthrombosis and macrothrombosis. This pilot study was designed to determine whether the angiographic presence or the extent of atherosclerosis was correlated with the activity of coagulationfactors in blood. A novel computational model was used to predict whether differences in the activity of coagulationfactors would alter the generation of thrombin. Blood was obtained for the determination of coagulationfactor activity from patients undergoing cardiac catheterization (n=50) who were grouped by the extent of their coronary artery disease (CAD). Generation of thrombin and factor (f) Xa were determined computationally. The activities of fII and fX were significantly lower in blood from patients with more severe CAD. Despite this, the time to clot (presumably reflecting a hypercoagulable state) was shorter in all patient groups than projected in healthy participants. Tissue factor pathway inhibitor concentrations were strongly associated with the generation of fXa and thrombin, and it is the best predictor of the time to clot. The balance between tissue factor and tissue factor pathway inhibitor appears to be a primary determinant of a hypercoagulable state that can accompany CAD. Lower concentrations of fX and fII in blood from patients with more severe CAD, who exhibit a shorter time to clot in vitro, are consistent with the clinical observation that patients at greater risk for thrombosis can also be at greater risk for bleeding.

The prevalence of serological markers for present and past hepatitis B virus (HBV) infection and antibodies against cytomegalovirus (CMV) among Norwegians with coagulationfactor defects was examined in serum samples collected before virus-inactivated coagulation concentrates came into use. Sera collected in 1985/86 from 324 of 377 (86%) registered persons with such defects were available. Three persons were chronic carriers of HBsAg. The prevalence of HBV antibodies was 28% compared with about 5% in the general population. The highest prevalence rate was found among patients with severe haemophilia A (44%) and in patients with haemophilia B (39%). The prevalence of anti-CMV antibodies was 75% which is similar to that found in the general Norwegian population.

Hereditary angioedema (HAE) is characterized clinically by recurrent acute skin swelling, abdominal pain, and potentially life-threatening laryngeal edema. Three forms of HAE have been described. The classic forms, HAE types I and II, occur as a consequence of mutations in the C1-inhibitor gene. In contrast to HAE types I and II, HAE type III has been observed exclusively in women, where it appears to be correlated with conditions of high estrogen levels—for example, pregnancy or the use of oral contraceptives. A recent report proposed two missense mutations (c.1032C→A and c.1032C→G) in F12, the gene encoding human coagulationfactor XII (FXII, or Hageman factor) as a possible cause of HAE type III. Here, we report the occurrence of the c.1032C→A (p.Thr328Lys) mutation in an HAE type III–affected family of French origin. Investigation of the F12 gene in a large German family did not reveal a coding mutation. Haplotype analysis with use of microsatellite markers is compatible with locus heterogeneity in HAE type III. To shed more light on the pathogenic relevance of the HAE type III–associated p.Thr328Lys mutation, we compared FXII activity and plasma levels in patients carrying the mutation with that of healthy control individuals. Our data strongly suggest that p.Thr328Lys is a gain-of-function mutation that markedly increases FXII amidolytic activity but that does not alter FXII plasma levels. We conclude that enhanced FXII enzymatic plasma activity in female mutation carriers leads to enhanced kinin production, which results in angioedema. Transcription of F12 is positively regulated by estrogens, which may explain why only women are affected with HAE type III. The results of our study represent an important step toward an understanding of the molecular processes involved in HAE type III and provide diagnostic and possibly new therapeutic opportunities. PMID:17186468

Hereditary angioedema (HAE) is characterized clinically by recurrent acute skin swelling, abdominal pain, and potentially life-threatening laryngeal edema. Three forms of HAE have been described. The classic forms, HAE types I and II, occur as a consequence of mutations in the C1-inhibitor gene. In contrast to HAE types I and II, HAE type III has been observed exclusively in women, where it appears to be correlated with conditions of high estrogen levels--for example, pregnancy or the use of oral contraceptives. A recent report proposed two missense mutations (c.1032C-->A and c.1032C-->G) in F12, the gene encoding human coagulationfactor XII (FXII, or Hageman factor) as a possible cause of HAE type III. Here, we report the occurrence of the c.1032C-->A (p.Thr328Lys) mutation in an HAE type III-affected family of French origin. Investigation of the F12 gene in a large German family did not reveal a coding mutation. Haplotype analysis with use of microsatellite markers is compatible with locus heterogeneity in HAE type III. To shed more light on the pathogenic relevance of the HAE type III-associated p.Thr328Lys mutation, we compared FXII activity and plasma levels in patients carrying the mutation with that of healthy control individuals. Our data strongly suggest that p.Thr328Lys is a gain-of-function mutation that markedly increases FXII amidolytic activity but that does not alter FXII plasma levels. We conclude that enhanced FXII enzymatic plasma activity in female mutation carriers leads to enhanced kinin production, which results in angioedema. Transcription of F12 is positively regulated by estrogens, which may explain why only women are affected with HAE type III. The results of our study represent an important step toward an understanding of the molecular processes involved in HAE type III and provide diagnostic and possibly new therapeutic opportunities.

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulationFactors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality

The changes of some factors of blood coagulation system in rats following frost-bite of both hind feet under hypoxia were investigated. Male Wistar rats weighed 200 +/- 20g were divided into four groups: normal control (C); frostbite at normoxia (FN); frostbite during acute hypoxia (FAH) and frostbite during hypoxia after altitude acclimation (FHAC). Bleeding time and clotting time, rate of clot-retraction, plasma content of 6-keto-PGF1 alpha and TXB2 were determined following exposure to cold. The results showed that bleeding time and clotting time were shortened, and rate of clot-retraction was decreased, plasma content of 6-keto-PGF1 alpha and TXB2, T/P ratio were increased significantly after exposure to cold in all frostbite groups, but these changes were more prominent in FHAC than those in FN and FAH. The results demonstrated that there were changes in blood coagulation system following cold injury, blood coagulability was increased. These changes were closely related to the degree of frostbite. In addition, the degree of cold injury was aggravated by altitude acclimation and this may play an important role in the pathological process of dysfunction leading to necrosis of local frostbite tissue.

Native Factor V is an anticoagulant, but when activated by thrombin, Factor X or platelet proteases, it becomes a procoagulant. Due to these double properties, Factor V plays a crucial role in the regulation of coagulation/anticoagulation balance. Factor V Leiden (FVL) disorder may lead to thrombophilia. Whether a reduction in the activation of Factor V or Factor V Leiden may correct the disposition to thrombophilia is unknown. Therefore we tested SV-IV Peptide 1–16 (i.e. a peptide derived by seminal protein vescicle number IV, SV-IV) to assess its capacity to inhibit the procoagulant activity of normal clotting factor V or Factor V Leiden (FVL). We found that SV-IV protein has potent anti-inflammatory and immunomodulatory properties and also exerts procoagulant activity. In the present work we show that the SV-IV Peptide 1–16, incubated with plasma containing normal Factor V or FVL plasma for 5 minutes reduces the procoagulant capacity of both substances. This is an anticoagulant effect whereas SV-IV protein is a procoagulant. This activity is effective both in terms of the coagulation tests, where coagulation times are increased, and in terms of biochemical tests conducted with purified molecules, where Factor X activation is reduced. Peptide 1–16 was, in the pure molecule system, first incubated for 5 minutes with purified Factor V then it was added to the mix of phosphatidylserine, Ca2+, Factor X and its chromogenic molecule Chromozym X. We observed a more than 50% reduction in lysis of chromogenic molecule Chromozym X by Factor Xa, compared to the sample without Peptide 1–16. Such reduction in Chromozym X lysis, is explained with the reduced activation of Factor X by partial inactivation of Factor V by Peptide 1–16. Thus our study demonstrates that Peptide 1–16 reduces the coagulation capacity of Factor V and Factor V Leiden in vitro, and, in turn, causes factor X reduced activation. PMID:18154667

The first line of host defense is the innate immune system that includes coagulationfactors and pattern recognition molecules, one of which is mannose-binding lectin (MBL). Previous studies have demonstrated that MBL deficiency increases susceptibility to infection. Several mechanisms are associated with increased susceptibility to infection, including reduced opsonophagocytic killing and reduced lectin complement pathway activation. In this study, we demonstrate that MBL and MBL-associated serine protease (MASP)-1/3 together mediate coagulationfactor-like activities, including thrombin-like activity. MBL and/or MASP-1/3 deficient hosts demonstrate in vivo evidence that MBL and MASP-1/3 are involved with hemostasis following injury. Staphylococcus aureus infected MBL null mice developed disseminated intravascular coagulation (DIC), which was associated with elevated blood IL-6 levels (but not TNF-α and multi-organ inflammatory responses). Infected MBL null mice also develop liver injury. These findings suggest that MBL deficiency may manifest into DIC and organ failure during infectious diseases. PMID:20399528

The present study reported the case of a Chinese boy who was diagnosed with Moyamoya disease (MMD) associated with Graves' disease (GD). An overactivation of von Willebrand factor (vWF) and coagulationfactor VIII (FVIII) was identified in the plasma of the patient. Thiamazole and metoprolol treatment was thus administrated. After 2 months of treatment, the patient's thyroid function returned to normal and the neurological symptoms improved gradually. At the same time, the activities of vWF and FVIII were depressed. During the 20-month follow-up, information regarding the neurological symptoms, cerebrovascular imaging, thyroid function, thyroid autoantibodies and coagulation parameters was collected. High levels of thyroid autoantibodies persisted throughout the follow-up period, while other coagulation parameters remained in the normal range. In conclusion, considering the vital role of vWF and FVIII in vascular diseases, it is hypothesized that these two factors may serve an important role in the occurrence of GD associated with MMD. PMID:27882137

The initiation of coagulation in trauma is thought to originate from exposed tissue factor (TF); recent data have led to the alternative hypothesis that damage-associated molecular patterns may contribute to postinjury coagulation. In acute traumatic coagulopathy, aberrant coagulation is mediated via the activated protein C (aPC) pathway; the upstream regulators of this process and its relation to TF remain uncharacterized. To examine the role of the TF pathway in mediating acute traumatic coagulopathy, we used specific antibody blockades in an established murine model of traumatic hemorrhagic shock, hypothesizing that both coagulation activation after injury and aPC-mediated coagulopathy are driven by TF via thrombin. Mice underwent an established model of trauma and hemorrhage and were subjected to either sham (vascular cannulation) or trauma-hemorrhage (cannulation, laparotomy, shock to mean arterial pressure of 35 mm Hg); they were monitored for 60 minutes before sacrifice. Mice in each group were pretreated with either targeted anti-TF antibody to block the TF pathway or hirudin for specific blockade of thrombin. Plasma was assayed for thrombin-antithrombin (TAT) and aPC by enzyme-linked immunosorbent assay. Compared with controls, trauma-hemorrhage mice treated with anti-TF antibody had significantly reduced levels of TAT (2.3 ng/mL vs. 5.7 ng/mL, p = 0.016) and corresponding decreases in aPC (16.3 ng/mL vs. 31.6 ng/mL, p = 0.034), with reductions to levels seen in sham mice. Direct inhibition of thrombin yielded similar results, with reduction in aPC to levels below those seen in sham mice. In this study, blockade of the TF pathway led to the attenuation of both thrombin production and aPC activation observed in traumatic shock. Specific thrombin inhibition achieved similar results, indicating that aPC-related coagulopathy is mediated via thrombin activated by the TF pathway. The near-complete blockade of TAT and aPC observed in this model argues for a

BACKGROUND The initiation of coagulation in trauma is thought to originate from exposed tissue factor (TF); recent data have led to the alternative hypothesis that DAMPs may contribute to post-injury coagulation. In acute traumatic coagulopathy (ATC), aberrant coagulation is mediated via the activated protein C (aPC) pathway; the upstream regulators of this process, and its relation to TF, remain uncharacterized. To examine the role of the TF pathway in mediating ATC, we employed specific antibody blockades in an established murine model of traumatic hemorrhagic shock, hypothesizing that both coagulation activation after injury and aPC-mediated coagulopathy are driven by TF via thrombin. METHODS Mice underwent an established model of trauma and hemorrhage, and were subjected to either sham (vascular cannulation), or trauma-hemorrhage (cannulation, laparotomy, shock to MAP 35mmHg); they were monitored for 60 min prior to sacrifice. Mice in each group were pre-treated with either targeted anti-TF antibody to block the TF pathway, or hirudin for specific blockade of thrombin. Plasma was assayed for thrombin-antithrombin (TAT) and aPC by ELISA. RESULTS Compared to controls, trauma-hemorrhage mice treated with anti-TFAb had significantly reduced levels of TAT (2.3 vs. 5.7 ng/mL, p=0.016), and corresponding decreases in aPC (16.3 vs. 31.6 ng/mL, p=0.034), with reductions to levels seen in sham mice. Direct inhibition of thrombin yielded similar results, with reduction in aPC to levels below those seen in sham mice. CONCLUSIONS In this study, blockade of the TF pathway led to attenuation of both thrombin production and aPC activation observed in traumatic shock. Specific thrombin inhibition achieved similar results, indicating that aPC-related coagulopathy is mediated via thrombin activated by the TF pathway. The near-complete blockade of TAT and aPC observed in this model argues for a dominant role of the TF-thrombin pathway in both coagulation activation after injury

Background: Various regions in Iran, especially the Khuzestan Province, have been covered by dust and dirt during the past two years due to environmental changes in the Middle East. We sought to evaluate the effect of these pollutants on the coagulantfactors of people residing in Abadan and Khoramshahr, two major cities of Khuzestan Province. Methods: One hundred twenty-nine healthy individuals were enrolled into this study, and their prothrombin time as well as fibrinogen, platelet, and Factor VIII levels were measured before and after climate changes. Results: After climate changes, the mean prothrombin time decreased, while the fibrinogen, platelet, and Factor VIII levels rose. Conclusion: The results of this study suggest that the pollutants deployed in the Middle East can affect prothrombin time as well as fibrinogen, platelet, and Factor VII levels considerably and increase coagulant state. The pollutants can, consequently, increase the risk of cardiovascular diseases. It seems that cooperation at government levels between Iran and its neighboring countries is required to reverse desertification and avoid inaccurate usage of subterranean water resources so as to lessen air pollution. PMID:23825886

Various regions in Iran, especially the Khuzestan Province, have been covered by dust and dirt during the past two years due to environmental changes in the Middle East. We sought to evaluate the effect of these pollutants on the coagulantfactors of people residing in Abadan and Khoramshahr, two major cities of Khuzestan Province. One hundred twenty-nine healthy individuals were enrolled into this study, and their prothrombin time as well as fibrinogen, platelet, and Factor VIII levels were measured before and after climate changes. After climate changes, the mean prothrombin time decreased, while the fibrinogen, platelet, and Factor VIII levels rose. The results of this study suggest that the pollutants deployed in the Middle East can affect prothrombin time as well as fibrinogen, platelet, and Factor VII levels considerably and increase coagulant state. The pollutants can, consequently, increase the risk of cardiovascular diseases. It seems that cooperation at government levels between Iran and its neighboring countries is required to reverse desertification and avoid inaccurate usage of subterranean water resources so as to lessen air pollution.

Summary Background Immunoglobulin (IG) products, including intravenous (IVIG) or subcutaneous (SCIG) immunoglobulins are considered safe and effective for medical therapy; however, a sudden and unexpected increase in thromboembolic events (TE) after administration of certain batches of IVIG products has been attributed to the presence of activated coagulationfactors, mainly factor XIa. Our aims were to examine the presence of enduring procoagulant activity during the manufacturing process of IGs, with special focus on monitoring factor XIa, and to evaluate the presence of in vitro procoagulant activity attributed to coagulationfactors in different lots of IVIG and SCIG. Methods Samples of different steps of IG purification, 19 lots of IVIG and 9 of SCIG were analyzed and compared with 1 commercial preparation of IVIG and 2 of SCIG, respectively. Factors II, VII, IX, XI and XIa and non-activated partial thromboplastin time (NAPTT) were assayed. Results The levels of factors II, VII, IX, X and XI were non-quantifiable once fraction II had been re-dissolved and in all analyzed lots of IVIG and SCIG. The level of factor XIa at that point was under the detection limits of the assay, and NAPTT yielded values greater than the control during the purification process. In SCIG, we detected higher concentrations of factor XIa in the commercial products, which reached values up to 5 times higher than the average amounts found in the 9 batches produced by UNC-Hemoderivados. Factor XIa in commercial IVIG reached levels slightly higher than those of the 19 batches produced by UNC-Hemoderivados. Conclusion IVIG and SCIG manufactured by UNC-Hemoderivados showed a lack of thrombogenic potential, as demonstrated not only by the laboratory data obtained in this study but also by the absence of any reports of TE registered by the post marketing pharmacovigilance department. PMID:26733772

Background: Testing coagulationfactor activities requires that multiple dilutions be assayed and analyzed to produce a single result. The slope of the line created by plotting measured factor concentration against sample dilution is evaluated to discern the presence of inhibitors giving rise to nonparallelism. Moreover, samples producing results on initial dilution falling outside the analytic measurement range of the assay must be tested at additional dilutions to produce reportable results. Methods: The complexity of this process has motivated a large clinical reference laboratory to develop advanced computer algorithms with automated reflex testing rules to complete coagulationfactor analysis. A method was developed for autoverification of coagulationfactor activity using expert rules developed with on an off the shelf commercially available data manager system integrated into an automated coagulation platform. Results: Here, we present an approach allowing for the autoverification and reporting of factor activity results with greatly diminished technologist effort. Conclusions: To the best of our knowledge, this is the first report of its kind providing a detailed procedure for implementation of autoverification expert rules as applied to coagulationfactor activity testing. Advantages of this system include ease of training for new operators, minimization of technologist time spent, reduction of staff fatigue, minimization of unnecessary reflex tests, optimization of turnaround time, and assurance of the consistency of the testing and reporting process. PMID:28706751

Procoagulant venoms of several Australian elapids contain proteinases that specifically activate prothrombin; among these, Group D activators are functionally similar to coagulationfactor Xa (FXa). Structural information on this class of prothrombin activators will contribute significantly towards understanding the mechanism of FXa-mediated prothrombin activation. Here we present the purification of Group D prothrombin activators from three Australian snake venoms (Hoplocephalus stephensi, Notechis scutatus scutatus and Notechis ater niger) using a single-step method, and their N-terminal sequences. The N-terminal sequence of the heavy chain of hopsarin D (H. stephensi) revealed that a fully conserved Cys-7 was substituted with a Ser residue. We therefore determined the complete amino acid sequence of hopsarin D. Hopsarin D shows approximately 70% similarity with FXa and approximately 98% similarity with trocarin D, a Group D prothrombin activator from Tropidechis carinatus. It possesses the characteristic Gla domain, two epidermal growth factor-like domains and a serine proteinase domain. All residues important for catalysis are conserved, as are most regions involved in interactions with factor Va and prothrombin. However, there are some structural differences. Unlike FXa, hopsarin D is glycosylated in both its chains: in light-chain residue 52 and heavy-chain residue 45. The glycosylation on the heavy chain is a large carbohydrate moiety adjacent to the active-site pocket. Overall, hopsarin D is structurally and functionally similar to mammalian coagulation FXa. PMID:12403650

Coagulationfactor IX (FIX) is an essential plasma protein for blood coagulation. The first epidermal growth factor (EGF) motif of FIX (EGF-F9) has been reported to attenuate cell adhesion to the extracellular matrix (ECM). The purpose of the present study was to determine the effects of this motif on cell adhesion and apoptosis. Treatment with a recombinant EGF-F9 attenuated cell adhesion to the ECM within 10 min. De-adhesion assays with native FIX recombinant FIX deletion mutant proteins suggested that the de-adhesion activity of EGF-F9 requires the same process of FIX activation as that which occurs for coagulation activity. The recombinant EGF-F9 increased lactate dehydrogenase (LDH) activity release into the medium and increased the number of cells stained with annexin V and activated caspase-3, by 8.8- and 2.7-fold respectively, indicating that EGF-F9 induced apoptosis. Activated caspase-3 increased very rapidly after only 5 min of administration of recombinant EGF-F9. Treatment with EGF-F9 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), but not that of phosphorylated MAPK 44/42 or c-Jun N-terminal kinase (JNK). Inhibitors of caspase-3 suppressed the release of LDH. Caspase-3 inhibitors also suppressed the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicated that EGF-F9 activated signals for apoptosis and induced de-adhesion in a caspase-3 dependent manner.

International Normalized Ratio (INR) measurements are used to monitor oral anticoagulation therapy with coumarins. Single coagulationfactor activities and calibrated automated thrombin (CAT) generation are considered as more advanced methods for evaluating overall haemostatic capacity. The aims were to assess the variability of INR, coagulationfactor activities, and CAT, during 24 h of storage of blood samples at ambient temperature. A total of 24 patients on stable coumarin treatment were followed prospectively for 6 weeks. INR was analyzed at 0, 6 and 24 h after blood sampling and 1-stage clotting activity of coagulationfactors II, VII, IX, and X as well as CAT generation was recorded after 0 and 24 h respectively. Statistical analyses included Bland-Altman plot, 95% limits of agreement, and a variability test using a mixed effect model. The level of INR remained statistically unchanged from 0 to 6 and 24 h of storage. Coagulationfactor activities and CAT revealed no significant difference induced by 24 h of storage, although the limits of agreement were wide. Patients' individual INR, coagulationfactor activities, and CAT generation were not significantly influenced by 24 h storage of blood samples, but for the CAT generation analyses a trend toward time dependency was detected.

Biological variation is usually estimated in healthy individuals during steady-state conditions. The aim of this study was to estimate the in-treatment biological variation of the International normalised ratio (INR) and to investigate to what extent the different levels of coagulationfactors could explain this variation. Blood samples were collected from randomly included patients on warfarin treatment. INR was determined on a laboratory instrument (STA Compact(®)) and on three point-of-care instruments (Simple Simon(®)PT, CoaguChek(®)XS and INRatio(™)). The level of fibrinogen, and the activity of coagulationfactors II, V, VII and X were determined. The in-treatment within- and between-subject coefficients of variation of INR were dependent on the method and varied between 18 and 24% and 13 and 19%, respectively, and were reduced to 3.9-5.1% and 2.3-5.8%, after correction for coagulationfactors which could explain 91-95% of the variance of INR. The in-treatment biological variation of INR was higher than reported for healthy individuals as well as patients in a steady-state condition, but by correcting for appropriate coagulationfactors it was reduced. The association between INR and coagulationfactors was different for the different PT methods mainly due to different sensitivity towards FII and FVII.

Bovine neonatal pancytopenia (BNP) is a newly emerging disease in many European countries that causes haemorrhagic diathesis and mortality in neonatal calves. This study tested the hypothesis that genetic factors might be involved in BNP, since genetic defects resulting in coagulation disorders have been described in many species, including cattle. A familial pattern of occurrence of BNP cases was observed in an experimental population of cattle in Germany and BNP was diagnosed in nine calves on an experimental dairy herd from May 2007 to December 2009. All affected calves were descendents of a single F(1) sire in a specific F(2) resource population generated from Charolais and German Holstein bloodlines. Sequence analysis of the bovine coagulationfactor XI (F11) gene as a functional candidate gene for BNP revealed an unusually high number of non-synonymous mutations within the gene compared to a whole genome mutation screen in cattle targetting random sequences. However, none of the mutations in the F11 gene were concordant with BNP status. Although these data and further pedigree analysis excluded a simple mode of inheritance of the BNP phenotype, there was a statistically significant (P=0.0001) accumulation of BNP cases in the specific pedigree examined, suggesting that a genetic component is involved in the development of BNP.

Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to β-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by β-factor C, in an LPS-dependent manner and that β-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C. PMID:26109069

CoagulationFactor (F)Xa is a serine protease that plays a crucial role during blood coagulation by converting prothrombin into active thrombin. Recently, however, it emerged that besides this role in coagulation, FXa induces intracellular signaling leading to different cellular effects. Here, we show that coagulationfactor (F)Xa drives tumor cells of epithelial origin, but not endothelial cells or monocytes, into apoptosis, whereas it even enhances fibroblast survival. FXa signals through the protease activated receptor (PAR)-1 to activate extracellular-signal regulated kinase (ERK) 1/2 and p38. This activation is associated with phosphorylation of the transcription factor CREB, and in tumor cells with up-regulation of the BH3-only pro-apoptotic protein Bim, leading to caspase-3 cleavage, the main hallmark of apoptosis. Transfection of tumor cells with dominant negative forms of CREB or siRNA for either PAR-1, Bim, ERK1 and/or p38 inhibited the pro-apoptotic effect of FXa. In fibroblasts, FXa-induced PAR-1 activation leads to down-regulation of Bim and pre-treatment with PAR-1 or Bim siRNA abolishes proliferation. We thus provide evidence that beyond its role in blood coagulation, FXa plays a key role in cellular processes in which Bim is the central player in determining cell survival.

Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)-Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulationfactor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulationfactor IX concentrate is less thrombogenic than factor IX complex.

The tissue factor (TF)/factor (F)VIIa complex is the primary initiator of coagulation in vivo. Tissue factor pathway inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Deficiencies of either TF or TFPI have not been reported in humans, and a complete absence of either of these two proteins in mice is embryonically lethal. To maintain normal hemostasis, levels of TF and TFPI need to be balanced. Increased levels of TF can overwhelm the inhibitory capacity of TFPI, resulting in thrombosis. Decreased levels of TF are associated with bleeding. Global assays of coagulation are defined as tests capable of evaluating all components of the clotting cascade that are present in plasma. In these tests the thrombogenic surface is either provided by platelets or exogenous phospholipids. Clotting assays currently used in clinical practice are not designed to measure endogenous levels of TF and TFPI. Therefore, there is a need to develop sensitive and specific assays for measuring levels of functional TF and TFPI in whole blood and plasma. These assays could be useful in patient management in many scenarios.

The tissue factor (TF)/factor (F)VIIa complex is the primary initiator of coagulation in vivo. Tissue factor pathway inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Deficiencies of either TF or TFPI have not been reported in humans, and a complete absence of either of these two proteins in mice is embryonically lethal. To maintain normal hemostasis, levels of TF and TFPI need to be balanced. Increased levels of TF can overwhelm the inhibitory capacity of TFPI, resulting in thrombosis. Decreased levels of TF are associated with bleeding. Global assays of coagulation are defined as tests capable of evaluating all components of the clotting cascade that are present in plasma. In these tests the thrombogenic surface is either provided by platelets or exogenous phospholipids. Clotting assays currently used in clinical practice are not designed to measure endogenous levels of TF and TFPI. Therefore, there is a need to develop sensitive and specific assays for measuring levels of functional TF and TFPI in whole blood and plasma. These assays could be useful in patient management in many scenarios. PMID:20978997

Global coagulation assays display variable effects at different concentrations of rivaroxaban. The aim of this study is to quantify the ex vivo effects of low-dose rivaroxaban on thrombophilia screening assays and coagulationfactor activities based on the administration time, and to show how to mask possible interferences. Plasma samples from 40 patients receiving rivaroxaban 10 mg daily were investigated to measure activities of clotting factor II, V, VII, VIII, IX, XI, XII and XIII; protein C- and protein S-levels; lupus anticoagulants; anticardiolipin IgG and IgM; D-dimer, heparin-platelet factor 4 (HPF4) antibodies and screening tests for von Willebrand disease (VWD). Two hours after rivaroxaban administration, the activities of clotting factors were significantly decreased to different extents, except for factor XIII. Dilution of plasma samples resulted in neutralisation of these interferences. The chromogenic protein C activity assay was not affected by rivaroxaban. Depending on the timing of tablet intake in relation to blood sampling protein S activity was measured falsely high when a clotting assay was used. False-positive results for lupus anticoagulants were observed depending on the assay system used and the administration time of rivaroxaban. ELISA-based assays such as anticardiolipin IgG and IgM, D-dimer, HPF4-antibodies and the turbidimetric assays for VWD were not affected by rivaroxaban. Specific haemostasis clotting tests should be performed directly prior to rivaroxaban intake. Assay optimisation in the presence of rivaroxaban can be achieved by plasma dilution. Immunologic assays are not influenced by rivaroxaban, while chromogenic assays can be used, when they do not depend on factor Xa.

Intracranial haemorrhage is a redoubtable complication during extracorporeal membrane oxygenation (ECMO) therapy. The underlying mechanisms of haemorrhagic diathesis are still not completely understood. This study was performed to evaluate a coagulation protocol for the regular analysis of acquired coagulation disorders and the systematic substitution of coagulationfactors to reach predefined target values. We hypothesised that using this strategy would lead to the identification of acquired bleeding disorders which cannot be monitored with standard coagulation tests and that substitution of the respective factors in a target-controlled approach could have an impact on the incidence and severity of intracranial haemorrhage. A protocol for the analysis of acquired coagulation disorders and the subsequent administration of associated factor concentrates was introduced. Previously, coagulation management was mainly based on clinical bleeding signs as the trigger for the administration of blood products. In this investigation, nineteen consecutive patients before (control group) and twenty consecutive patients after the implementation of the protocol (intervention group) have been included in the study. Eighty-eight percent of the patients developed factor XIII deficiency, 79% acquired von Willebrand syndrome, 40% fibrinogen deficiency and 54% of the patients showed a decline in platelet count >20% within the first 24 hours of ECMO therapy. In 6 out of 19 (31%) patients in the control group and in 2 patients out of 20 (10%) in the intervention group, intracranial haemorrhage was detected. Whilst 5 of 6 patients in the control group died because of fatal bleeding, both of the patients in the intervention group recovered with a favourable neurologic outcome. Veno-venous ECMO therapy leads to thrombocytopenia, factor XIII and fibrinogen deficiency as well as acquired von Willebrand syndrome. The implementation of a coagulation protocol including a standardized

Two different lipophilic photoreagents, (/sup 3/H)adamantane diazirine and 3-(trifluoromethyl)-3-(m-(/sup 125/I)iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulationfactor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. Methods and Results Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. Conclusions Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP. PMID:27764259

Blood coagulationfactor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.

Three activated partial thromboplastin time (APTT) reagent test systems, General Diagnostics Automated APTT, American Dade Actin FS, and Pacific Hemostasis (Thromboscreen KAPTT) reagent, containing different activators for the APTT assay, were evaluated for their precision and sensitivity to factor deficiencies in the intrinsic coagulation system. The data suggest that micronized silica and ellagic acid reagent systems were similar in sensitivity to Factor VIII, X, and XII deficiencies, whereas, the micronized kaolin reagent was significantly less sensitive to these deficiencies. Factor XI deficiency was detected equally well with the use of all three reagent systems. The ellagic acid reagent was somewhat more sensitive to Factor IX deficiency than the micronized silica reagent, and the micronized kaolin reagent was again least sensitive. Both the micronized silica and ellagic acid based reagents were insensitive to all but severe deficiencies in prekallikrein, whereas the micronized kaolin reagent was unable to detect this deficiency. All three reagents were insensitive to all but severe deficiencies in high-molecular-weight kininogen. The authors conclude that the reagent systems tested, containing micronized silica or ellagic acid as activators, are similar in sensitivity when used in a routine activated partial thromboplastin time to screen for factor deficiencies, whereas the reagent system containing micronized kaolin as an activator is less sensitive.

To identify potential mutations and explore the molecular mechanism underlying combined inherited coagulationfactors VII(FVII) and X(FX) deficiency for a family featuring consanguineous marriage between maternal cousins. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, FVII activity (FVII:C), FX activity (FX:C), FVII antigen (FVII:Ag), FX antigen (FX:Ag) and other coagulant parameters of the proband and 5 family members were measured. Potential mutations in exons, exon-intron boundaries and 5', 3' untranslated sequences of F7 and F10 genes were screened by polymerase chain reaction and direct sequencing. Suspected mutations were confirmed by sequencing the opposite strand. PT and APTT of the proband were obviously prolonged to become 76.4 s and 60.2 s, respectively. FVII:C, FVII:Ag,FX:C and FX:Ag of the proband were obviously reduced to become 4%, 6%, 6% and 33%, respectively. Both PT and APTT of her grandmother, father, mother and daughter were slightly prolonged, which have measured 16.4 s, 15.8 s,16.9 s, 16.5 s, and 44.0 s, 42.1 s, 41.1 s, 43.5 s, respectively. And their FVII:C (34%, 39%, 31%, 40%, respectively), FX:C (50%, 58%, 47%, 42%, respectively) and FX:Ag (51%, 54%, 58%, 47%, respectively) were slightly reduced, while FVII:Ag was in the normal range. The coagulant parameters of her younger brother were within normal range. Two homozygous mutations, g.11267C to T in exon 8 of F7 gene, which resulted in an Arg277Cys substitution, and g.28139G to T in exon 8 of F10 gene which led to a Val384Phe substitution, were identified in the proband. The proband's grandmother, parents and daughter were heterozygous for both Arg277Cys and Val384Phe mutationss. Wild-type alleles of both F7 and F10 genes were also found in the younger brother. A homozygous Arg277Cys mutation and a Val384Phe mutation have been respectively identified in the F7 and F10 genes, which can explain the low levels of FVII and FX in this family. The former has been

Objective To identify preoperative and intraoperative factors and conditions that predicts the beneficial application of a high-frequency electromagnetic field (EMF) system for tumor vaporization and coagulation. Methods One hundred three subsequent patients with brain tumors were microsurgically treated using the EMF system in addition to the standard neurosurgical instrumentarium. A multivariate analysis was performed regarding the usefulness (ineffective/useful/very helpful/essential) of the new technology for tumor vaporization and coagulation, with respect to tumor histology and location, tissue consistency and texture, patients' age and sex. Results The EMF system could be used effectively during tumor surgery in 83 cases with an essential contribution to the overall success in 14 cases. In the advanced category of effectiveness (very helpful/essential), there was a significant difference between hard and soft tissue consistency (50 of 66 cases vs. 3 of 37 cases). The coagulation function worked well (very helpful/essential) for surface (73 of 103 cases) and spot (46 of 103 cases) coagulation when vessels with a diameter of less than one millimeter were involved. The light-weight bayonet hand piece and long malleable electrodes made the system especially suited for the resection of deep-seated lesions (34 of 52 cases) compared to superficial tumors (19 of 50 cases). The EMF system was less effective than traditional electrosurgical devices in reducing soft glial tumors. Standard methods where also required for coagulation of larger vessels. Conclusion It is possible to identify factors and conditions that predict a beneficial application of high-frequency electromagnetics for tumor vaporization and coagulation. This allows focusing the use of this technology on selective indications. PMID:18445296

Androgen receptor (AR) is widely expressed in breast cancer; however, there is limited information on the key molecular functions and gene targets of AR in this disease. In this study, gene expression data from a cohort of 52 breast cancer cell lines was analyzed to identify a network of AR co-expressed genes. A total of three hundred genes, which were significantly enriched for cell cycle and metabolic functions, showed absolute correlation coefficients (| CC |) of more than 0.5 with AR expression across the dataset. In this network, a subset of 35 “AR-signature” genes were highly co-expressed with AR (| CC| > 0.6) that included transcriptional regulators PATZ1, NFATC4, and SPDEF. Furthermore, gene encoding coagulationfactor VII (F7) demonstrated the closest expression pattern with AR (CC= 0.716) in the dataset and factor VII protein expression was significantly associated to that of AR in a cohort of 209 breast tumors. Moreover, functional studies demonstrated that AR activation results in the induction of factor VII expression at both transcript and protein levels and AR directly binds to a proximal region of F7 promoter in breast cancer cells. Importantly, AR activation in breast cancer cells induced endogenous factor VII activity to convert factor X to Xa in conjunction with tissue factor. In summary, F7 is a novel AR target gene and AR activation regulates the ectopic expression and activity of factor VII in breast cancer cells. These findings have functional implications in the pathobiology of thromboembolic events and regulation of factor VII/tissue factor signaling in breast cancer. PMID:25447311

Summary Background Upon contact with an appropriate surface, factor XII (FXII) undergoes autoactivation or cleavage by kallikrein. Zn2+ is known to facilitate binding of FXII and the cofactor, high molecular weight kininogen (HK), to anionic surfaces. Objectives To investigate whether transition metals immobilized on liposome surfaces can initiate coagulation via the contact pathway. Methods & Results Liposomes containing a metal ion-chelating lipid (DOGS-NTA) were prepared by membrane extrusion (20% DOGS-NTA, 40% phosphatidylcholine, 10% phosphatidylserine, and 30% phosphatidylethanolamine). Ni2+ immobilized on such liposomes accelerated clotting in normal, but not FXI- or FXII-deficient plasma. Results were comparable to a commercial aPTT reagent. Charging such liposomes with other transition metals revealed differences in their procoagulant capacity, with Ni2+> Cu2+> Co2+ and Zn2+. Plasma could be depleted of FXI, FXII and HK by adsorption with Ni2+-containing beads, resulting in delayed clot times. Consistent with this, FXI, FXII and HK bound to immobilized Ni2+ or Cu2+ with high affinity as determined by surface plasmon resonance. In the presence of Ni2+-bearing liposomes, Km and kcat values derived for autoactivation of FXII and prekallikrein, as well as for activation of FXII by kallikrein or prekallikrein by FXIIa, were similar to literature values in the presence of dextran sulfate. Conclusions Immobilized Ni2+ and Cu2+ bind FXII, FXI and HK with high affinity and stimulate activation of the contact pathway, driving FXII-mediated coagulation. Activation of the contact system by immobilized transition metals may have implications during pathogenic infection or in individuals exposed to high levels of pollution. PMID:22905925

The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a wall shear rate of 57, 300, or 1130 sec{sup {minus}1}. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2{endash}-to 3{endash}fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminated by the lipoprotein-associated coagulation inhibitor. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.

Despite administration of vitamin K (VK), some infants show lower activity of VK-dependent coagulationfactors and they could develop intracranial hemorrhage. For preventing VK deficiency bleeding (VKDB) in infants, oral administration of VK and a screening test for VK deficiency are carried out in Japan. For the screening, the total activity of VK-dependent coagulationfactors is measured using a commercial product, Normotest. This study was undertaken to clarify the importance of the following genetic and environmental factors on the coagulation status in one-month-old infants: two polymorphisms in the factor VII gene, -323P0/10 (a 10-bp insertion in the promoter region at position -323) and R353Q (the replacement of arginine [R] with glutamine [Q] at residue 353) and sex, age, gestational age, birth weight, and feeding regimen. Two hundred Japanese infants (34.6 +/- 4.0 days old) were screened for VK-dependent coagulation activity with Normotest and were genotyped for the two polymorphisms. Among the subjects screened, 18 infants (9%) carried the P10 allele and 26 (13%) carried the R353Q allele. Multiple regression analysis showed that the 10-bp inserted (P10) allele or the Q allele was associated with the lower coagulation activities. The coagulation activities for the R/Q genotype were significantly lower than those for the R/R genotype and those for the P0/P10 genotype were significantly lower than those for the P0/P0 genotype. Therefore, infants who carry the P10 allele or the Q allele show lower activity of VK-dependent coagulationfactors. These infants may have a higher risk of VKDB manifestation.

Terminal dry heat treatment effectively inactivated hepatitis A virus (HAV) and canine parvovirus added to high-purity factor VIII. After 24 h at 80 degrees C, HAV infectivity was reduced by > or = 4.3 log10 TCID50, as measured in a newly developed infectivity assay. The same reduction in virus titer was achieved after 2 h and before 6 h at 90 degrees C. Inactivation of hepatitis A virus was also seen in the freeze-drying step prior to heat treatment with an approximately 2.0 log10 reduction in titer. Similar results were obtained with a high-purity factor IX concentrate. Canine parvovirus was also inactivated at both temperatures, with residual infectivity being undetected after 48 h at 80 degrees C or 10 h at 90 degrees C. Canine parvovirus was not affected by lyophilisation. Canine parvovirus measurements by PCR did not reflect the levels of infectivity measured by the tissue-culture-based method. The addition of the terminal dry heat treatment to solvent/detergent could effectively eliminate the potential contamination of solvent/detergent-treated coagulationfactor concentrates by non-lipid-enveloped viruses. However, careful evaluation for any increased induction of non-antigens for factor VIII, as a consequence of such treatment, is needed before use in patients can be recommended.

This study aims to investigate the molecular mechanisms underlying the pathogenesis of severe acute pancreatitis (SAP) and SAP-associated liver injury, we performed an association analysis of the functions of tissue factor (TF) and blood coagulation system in both SAP patients and mouse SAP model. Our results showed that serum TF and tissue factor-microparticle (TF-MP) levels were highly up-regulated in both SAP patients and SAP mouse model, which was accompanied by the dysfunction of blood coagulation system. Besides, TF expression was also highly up-regulated in the Kupffer cells (KCs) of SAP mouse model. After inhibiting KCs in SAP mouse model, the amelioration of blood coagulation system functions was associated with the decrease in serum TF and TF-MPs levels, and the reduction of SAP-associated liver injury was associated with the decrease of TF expression in KCs. In conclusion, the dis-regulated TF expression and associated dysfunction of blood coagulation system are critical factors for the pathogenesis of SAP and SAP-associated liver injury. TF may serve as a potential and effective target for treating SAP and SAP-associated liver injury.

Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulationfactors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

The coagulationfactor XIII(FXIII) is a plasma circulating heterotetrameric protransglutaminase that acts at the end of the coagulation cascade by covalently cross-linking preformed fibrin clots (to themselves and to fibrinolytic inhibitors) in order to stabilize them against fibrinolysis. It circulates in the plasma as a heterotetramer composed of two homomeric catalytic Factor XIIIA2 (FXIIIA2) and two homomeric protective/carrier Factor XIIIB2 subunit (FXIIIB2). Congenital deficiency of FXIII is of two types: severe homozygous/compound heterozygous FXIII deficiency which results in severe bleeding symptoms and mild heterozygous FXIII deficiency which is associated with mild bleeding (only upon trauma) or an asymptomatic phenotype. Defects in the F13B gene (Factor XIIIB subunit) occur more frequently in mild FXIII deficiency patients than in severe FXIII deficiency. We had recently reported secretion-related defects for seven previously reported F13B missense mutations. In the present study we further analyze the underlying molecular pathological mechanisms as well as the heterozygous expression phenotype for these mutations using a combination of in vitro heterologous expression (in HEK293T cells) and confocal microscopy. In combination with the in vitro work we have also performed an in silico solvated molecular dynamic simulation study on previously reported FXIIIB subunit sushi domain homology models in order to predict the putative structure-functional impact of these mutations. We were able to categorize the mutations into the following functional groups that: (1) affect antigenic stability as well as binding to FXIIIA subunit, that is, Cys5Arg, Cys316Phe, and Pro428Ser (2) affect binding to FXIIIA subunit with little or no influence on antigenic stability, that is, Ile81Asn and Val401Gln c) influence neither aspects and are most likely causality linked polymorphisms or functional polymorphisms, that is, Leu116Phe and Val217Ile. The Cys5Arg mutation was the

To evaluate and characterize the certified reference materials for coagulationfactor Ⅷ (FⅧ) and factor Ⅸ (FⅨ) activity testing. The homogeneity and stability of three lots of certified reference materials (F01-F03) with different factor concentrations were evaluated according to guidelines"Reference materials-general and statistical principles for certification","Guidance on evaluating the homogeneity and stability of samples used for proficiency testing"and"Technical Norm of Primary Reference Material". The certified reference materials were characterized by eight laboratories using one-stage method, which were calibrated by the coagulation standard provided by the National Institute for Biological Standards and Control (NIBSC) in UK. The Coefficient of Variation (CV) of homogeneity test of FⅧ activity of three lots of certified reference materials were 3.9%, 3.3% and 3.4%, respectively. While that of FⅨ activity were 3.7%, 3.0% and 1.8%, respectively. The results of one-way ANOVA showed that all certified reference materials had good homogeneity (P>0.05), and the between-bottle homogeneity uncertainties (ubb) of FⅧ and FⅨ activity were 0.5%-2.9% and 0.1%-3.9%, respectively. All certified reference materials stored in -80 ℃ remained stable in 9 months by trend analysis, and the long-term stability uncertainties(ults) of FⅧ and FⅨ activity were 0.5%-5.1% and 1.3%-4.4%, respectively. The characterization uncertainties (uchar) of FⅧ and FⅨ activity testing were 0.9%-2.4% and 1.1%-2.4%, respectively. The combined uncertainties and extended uncertainties (coverage factor k=2) were calculated. The assigned values of each lot of certified reference materials for FⅧ activity were (85±13)%, (36.0±3.4)% and (20.5±2.3)%, and that were (102±13)%, (47.8±6.9)% and (29.3±3.8)% for FⅨ activity, respectively. The certified reference materials for FⅧ and FⅨ activity testing have good homogeneity and stability. The results of the

The prophylactic treatment of haemophilia B and the management of haemophilia A or B with inhibitors demand frequent administrations of coagulationfactors due to the suboptimal half-lives of the products commercially available and currently in use, e.g. recombinant factor IX (rFIX) and recombinant factor VIIa (rFVIIa), respectively. The extension of the half-lives of rFIX and rFVIIa could allow for longer intervals between infusions and could thereby improve adherence and clinical outcomes and may improve quality of life. Albumin fusion is one of a number of different techniques currently being examined to prolong the half-life of rFIX and rFVIIa. Results from a phase I clinical trial demonstrated that the recombinant fusion protein linking FIX to albumin (rIX-FP) has a five-times longer half-life than rFIX, and preclinical studies with the recombinant fusion protein linking FVIIa to albumin (rVIIa-FP) suggest that rVIIa-FP possesses a significantly extended half-life versus rFVIIa. In this review, we describe albumin fusion technology and examine the recent progress in the development of rIX-FP and rVIIa-FP.

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulationfactors Xa (FXa) and XIa. Here we show that ZPI and its cofactor, protein Z (PZ), inhibit procoagulant membrane-bound factor Xa by the branched pathway acyl-intermediate trapping mechanism used by other serpins, but with significant variations of this mechanism that are unique to ZPI. Rapid kinetic analyses showed that the reaction proceeded by the initial assembly of a membrane-associated PZ-ZPI-FXa Michaelis complex (K(M) 53+/-5 nM) followed by conversion to a stable ZPI-FXa complex (k(lim) 1.2+/-0.1 s(-1)). Cofactor premixing experiments together with independent kinetic analyses of ZPI-PZ and factor Xa-PZ-membrane complex formation suggested that assembly of the Michaelis complex through either ZPI-PZ-lipid or factor Xa-PZ-lipid intermediates was rate-limiting. Reaction stoichiometry analyses and native PAGE showed that for every factor Xa molecule inhibited by ZPI, two serpin molecules were cleaved. Native PAGE and immunoblotting showed that PZ dissociated from ZPI once ZPI forms a stable complex with FXa, and kinetic analyses confirmed that PZ acted catalytically to accelerate the membrane-dependent ZPI-factor Xa reaction. The ZPI-FXa complex was only transiently stable and dissociated with a rate constant that showed a bell-shaped pH dependence indicative of participation of factor Xa active-site residues. The complex was detectable by SDS-PAGE when denatured at low pH, consistent with it being a kinetically trapped covalent acyl-intermediate. Together our findings show that ZPI functions like other serpins to regulate the activity of FXa but in a manner uniquely dependent on protein Z, procoagulant membranes, and pH.

FFP is considered adequate for transfusion up to 24 hours after thawing and is currently used most often to replace deficient clotting factors, such as in warfarin overdose. We set to examine the levels of vitamin K-dependent factors (i.e., prothrombin, FVII, F IX, FX), as well as fibrinogen, upon twice freezing and thawing of FFP. If factor levels in refrozen FFP remain within normal limits, this component can possibly be transfused, thus avoiding wastage of precious blood components. Twenty units of FFP, five units of each blood group A, B, AB, and O, were thawed, and aliquots were taken for measurement of coagulationfactors. The plasma units were then kept for 24 hours at 4 degrees C, at which point a second aliquot was taken, The remaining FFP units were refrozen and kept at -80 degrees C for 1 week. The above procedure was then repeated. Coagulation-factor activity and fibrinogen level were measured by the coagulation analyzer. The mean levels of prothrombin, FVII, F IX, FX, and fibrinogen of each blood group (A, B, AB, and O) were calculated for each of four time points and found not statistically different (p > 0.05). Therefore, the rest of the analysis was done for all 20 FFP units as one group. The mean +/- SD levels of each coagulationfactor at each time point demonstrated that all levels were within normal limits of all factors measured and that for none of the factors was there a significant decay of activity. The levels of prothrombin, FVII, F IX, FX, and fibrinogen remain stable and adequate for transfusion in twice-thawed-and-refrozen FFP. This component can be safely used for transfusion as a source of vitamin K-dependent clotting factors and fibrinogen.

Exposure of blood to tissue factor leads to the formation of a high affinity tissue factor/factor VIIa complex which initiates blood coagulation. As a first step toward obtaining structural information of this enzyme system, a complex of active-site inhibited factor VIIa (F.VIIai) and soluble tissue factor (sTF) was prepared for crystallization. Crystals were obtained, but only after long incubation times. Analysis by SDS-PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N- and C-terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 x 0.12 x 0.6 mm and showed diffraction to a resolution of 3 A.

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

: The role of coagulationfactor XI (FXI) in the cause of arterial thrombotic events remains uncertain. We examined the association of FXI with incident coronary heart disease (CHD), ischemic stroke, and hemorrhagic stroke. Data were from 3394 adults (mean age: 74.5 years) enrolled in the Cardiovascular Health Study who had FXI antigen from plasma samples drawn in 1992-1993 and were followed for cardiovascular events until 30 June 2013. Approximately 63% of participants were women and 17% were black. FXI levels were higher in blacks and women, showed positive associations with high-density lipoprotein and total cholesterol, BMI and diabetes, and negative associations with age and alcohol intake. During median follow-up of 13 years, we identified 1232 incident CHD, 473 ischemic stroke, and 84 hemorrhagic stroke events. In multivariable Cox models adjusted for traditional cardiovascular disease risk factors, the hazard ratio per one SD (32.2 mg/dl) increment of FXI was 1.02 (95% confidence interval: 0.96-1.08) for CHD; 0.94 (0.85-1.04) for ischemic stroke, and 0.85 (0.65-1.10) for hemorrhagic stroke. In this prospective cohort of elderly adults, there was no statistically significant association of higher FXI levels with incident CHD and stroke.

Immoderate blood clotting constitutes a risk factor for cardiovascular disease in modern industrialised societies, but is believed to have conferred a survival advantage, i.e. faster recovery from bleeding, on our ancestors. Here, we investigate the evolutionary history of the CoagulationFactor VII gene (F7) by analysing five cardiovascular-risk-associated mutations from the F7 promoter and nine neutral polymorphisms (six SNPs and three microsatellites) from the flanking region in 16 populations from the broader Mediterranean region, South Saharan Africa and Bolivia (687 individuals in total). Population differentiation and selection tests were performed and linkage disequilibrium patterns were investigated. In all samples, no linkage disequilibrium between adjacent F7 promoter mutations -402 and -401 was observed. No selection signals were detected in any of the samples from the broader Mediterranean region and South Saharan Africa, while some of the data suggested a potential signal of positive selection for the F7 promoter in the Native American samples from Bolivia. In conclusion, our data suggest, although do not prove, different evolutionary histories in the F7 promoter region between Mediterraneans and Amerindians.

Coronary heart disease (CHD) is the leading cause of mortality and morbidity in the United States. Currently, there are approximately 12 million Americans with CHD, which is most frequently caused by atherosclerosis. The thrombotic complications of atherosclerosis, such as acute coronary syndrome and ischemic stroke, can be fatal and those who survive such events have a far greater risk of future cardiovascular events. This huge medical need cries out for improved novel anticoagulants, antiplatelet agents, and profibrinolytic agents. These agents will successfully respond to the medical need by providing safe, effective, and easily administered treatments that have little, if any, drug and food interactions and that require minimal monitoring. The currently approved antiplatelet agent, clopidogrel, has satisfied some of these requirements and has played a large role in expanding the antithrombotic market over the past few years. New antithrombotics approaching the marketplace, such as the prodrug thrombin inhibitor ximelagatran, have promise in expanding the antithrombotic market further. Over the past two decades, the pharmaceutical industry has mounted a huge effort to develop antithrombotics that function by inhibiting key enzymes positioned at "higher" levels of the coagulation system. Direct inhibitors of factor Xa, which may provide a better safety and efficacy profile than currently available agents, appear to be the next major class of antithrombotic agents poised to take the pharmaceutical industry one step closer to delivering the ideal antithrombotic agent. This review focuses on recent innovations in the discovery and development of potent parenteral and oral direct factor Xa inhibitors.

Bovine Factor X is a zymogen involved in blood coagulation that is converted to activated Factor X in the presence of Ca(II) by the coagulant protein of Russell's viper venom. To monitor structural transitions in Factor X during conversion to activated Factor X, the ultraviolet absorption, fluorescence emission, and circular dichroism spectra of activated Factor X and Factor X were compared. The ultraviolet absorption difference spectrum in the aromatic region comparing activated Factor X and Factor X indicates minima at 292.5, 285, and 278 nm and a small maximum at 305 nm; these differences are due to tryptophan and tyrosine perturbations. The activation of Factor X at 25 degrees in the presence of 8.3 mM CaCl2 with the use of Factor X:venom coagulant protein in molar rations of 1500:1 yielded a time-dependent increase in this spectrum which was linear for about 60 min and which temporally paralleled the development of activated Factor X activity. The binding of Ca(II) to factor X or activated Factor X is associated with a red-shifted tryptophan difference spectrum; however, this perturbation appears to make only a small contribution to the total perturbation observed during Factor X activation. Solvent perturbation studies in 20% glycerol suggest that an average of 3.1 tryptophan residues and 9.0 tyrosine residues are exposed to solvent in Factor X in 8.3 mM CaCl2 at pH 7.4; an additional 0.5 tryptophan residue and tyrosine reside become exposed to solvent during activation of Factor X in 8.3 mM CaCl2. The activation of Factor X by the venom coagulant protein is associated with a small red shift in the intrinsic tryptophan fluorescence emission spectrum. Far- and near-ultraviolet circular dichroism spectroscopy detected no difference between Factor X and activated Factor X. In summary, the activation of Factor X to activated Factor X appears associated with exposure of tryptophan and tyrosine side chains previously buried within the protein and with minimal

Removal of leucocytes from blood products, namely leucoreduction, improves the safety of blood transfusion by reducing adverse events associated with the incidental transfusion of leucocytes. Coagulationfactors might be compromised during leucoreduction because of exposure of plasma to a variety of filter materials. The aim of the current study was to assess the effect of different methods of prestorage leucofiltration (apheresis and whole blood filters) on prothrombin time, international normalized ratio, partial thromboplastin time and factors V and VIII. There was a significant prolongation of prothrombin time as well as elevation of international normalized ratio in plasma after leucoreduction (14.5 ± 0.7 s vs. 13.9 ± 0.7 s, P = 0.008 and 1.14 ± 0.07 vs. 1.09 ± 0.07, P = 0.005, respectively). Also, there was a statistically significant prolongation of activated partial thromboplastin time in nonleucoreduced plasma (55.6 ± 9.9 s vs. 43.2 ± 12.8 s, P = 0.001). There was no significant filtration effect on factors V and VIII levels. Furthermore, there was no significant difference in factors V and VIII levels between plasma filtered by inline whole blood filters and apheresis machine. Leucodepleted plasma originating from both inline whole blood filter and apheresis machine maintained satisfactory levels of factors V and VIII.

The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX. Initial rates of trichloroacetic acid-soluble /sup 3/H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl/sub 2/ (5 mM) and with pure (greater than 98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of /sup 3/H preceded the appearance of Factor IXa activity, and the percentage of /sup 3/H released remained constant when the mole fraction of /sup 3/H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r greater than 0.98, P less than 0.001) was observed between initial rates of /sup 3/H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include K/sub m/ (0.49 microM) of about five- to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (k/sub cat/) (7.7/s) is approximately 20-50 times higher than that reported by Zur and Nemerson for Factor IX activation by Factor VIIa plus tissue factor. Therefore, depending on the relative amounts of Factor XIa and Factor VIIa generated in vivo and other factors which may influence reaction rates, these kinetic parameters provide part of the information required for assessing the relative contributions of the intrinsic and extrinsic pathways to Factor IX activation, and suggest that the Factor XIa catalyzed reaction is physiologically significant.

To explore the role of small-dose recombinant human coagulationfactor VIIa (rFVIIa) for coagulopathy in patients with isolated traumatic brain injury. A total of 86 isolated traumatic brain patients with coagulopathy were treated at our neurosurgery intensive care unit (NICU) from January 2010 to December 2012. Their trauma registry data included mortality, pre-and post-rFVIIa coagulation parameters. Two-tailed paired t-test was used to determine significant changes in coagulation parameters and other major clinical parameters. Twenty-seven patients made up the low-dose rFVIIa (20 µg/kg) group. And the control group had 59 well-matched subjects. At admission, age, blood pressure, Glasgow coma scale score, hemoglobin, platelets and international normalize ratio were similar in both groups. After treatment, the INR of patients on rFVIIa was lower than that of the conventional treatment group (1.1 ± 0.2 vs 1.2 ± 0.2, P < 0.01) and it declined more in the rFVIIa group (0.3 ± 0.2 vs 0.1 ± 0.4, P = 0.05). No significant difference existed in mortality or length of stay between two groups.There was no occurrence of subsequent thromboembolic events. The application of small-dose rFVIIa can effectively reduce the value of INR and improve the coagulation status of patients. During the course of treatment, no major adverse events occur.

The growth rate and size of floc formation is of great importance in water treatment especially in coagulation process. The floc formation kinetics and the coagulation efficiency of synthetic water were investigated by using an on-line continuous optical photometric dispersion analyze and the analysis of water quality. Experimental conditions such as alum dosage, pH value for coagulation, stirring intensity and initial turbidity were extensively examined. The photometric dispersion analyze results showed that coagulation of kaolin suspensions with two coagulants (alum and polyaluminium chloride) could be taken as a two-phase process: slow and rapid growth periods. Operating conditions with higher coagulant doses, appropriate pH and average shear rate might be particularly advantageous. The rate of overall floc growth was mainly determined by a combination of hydraulic and water quality conditions such as pH and turbidity. The measurement of zeta potential indicates that polyaluminium chloride exhibited higher charge-neutralizing ability than alum and achieved lower turbidities than alum for equivalent Al dosages. Under the same operating conditions, the alum showed a higher grow rate, but with smaller floc size.

Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulationfactor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5–FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad–FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy. PMID:27014840

Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulationfactor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5-FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad-FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy.

Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes.

Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12–/– mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes. PMID:26193639

Hemophilia B is an X-linked recessive bleeding disorder caused by abnormalities of the coagulationfactor IX gene (F9). Insertion mutations in F9 ranging from a few to more than 100 base pairs account for only a few percent of all hemophilia B cases. We investigated F9 to elucidate genetic abnormalities causing severe hemophilia B in a Japanese subject. We performed PCR-mediated analysis of F9 and identified a large insertion in exon 6. Next, we carried out direct sequencing of a PCR clone of the whole insert using nested deletion by exonuclease III and S1 nuclease. We identified an approximately 2.5-kb SINE-VNTR-Alu (SVA)-F element flanked by 15-bp duplications in the antisense orientation in exon 6. Additionally, we carried out exontrap analysis to assess the effect of this retrotransposition on mRNA splicing. We observed that regular splicing at exons 5 and 6 of F9 was disturbed by the SVA retrotransposition, suggesting that abnormal FIX mRNA may be reduced by nonsense-mediated mRNA decay. In conclusion, this is the first report of SVA retrotransposition causing severe hemophilia B; only five cases of LINE-1 or Alu retrotranspositions in F9 have been reported previously.

Objectives: In the current study, we compared the effects of low- and high-flow anesthesia techniques on hemorheology and coagulation parameters in patients who received sevofluran. Methods: Forty patients classified as Risk Group I–II according to American Society of Anesthesiologists’ (ASA) guidelines who were scheduled to undergo general anesthesia were randomly assigned to one of two groups. Low-flow anesthesia was administered to the first group, and high-flow anesthesia was used in the second group. Blood samples were obtained in the preoperative and peroperative periods (at 60 and 120 min) for determination of blood and plasma viscosity, plasma oncotic pressure, international normalized ratio (INR), phorotrombin time (PT), activated partial phorotrombin time (aPTT) and fibrinogen. Blood was also drawn for analysis of factor VIII (FVIII) activity, which was measured in the preoperative period and at postoperative six hour. Results: The peroperative plasma viscosity was significantly low in Group 1 relative to Group 2. aPTT was significantly elevated at 60 minutes in Group 1 relative to Group 2, but the increase at 120 minutes was not significant. Conclusion: The effects of low-flow anesthesia on hemorheology were greater than those of high-flow anesthesia. PMID:26150868

A purification process to obtain coagulationfactor VIII (F VIII) with high activity from human plasma was established. Based on the analysis of the size ratio between F VIII and matrix porous medium and its effect on the protein activity, a novel purification process designed was superporous ion exchange chromatography (IEC). The operating conditions of gigaporous and traditional anion exchange chromatography were optimized separately. The chromogenic substrate, gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to monitor the bioactivity and purity of the chromatographic products. The results showed that the superporous medium could not only protect structure of macro-protein but also enhance its mass transfer, finally giving FVIII product with high activity. The yield of F VIII in superporous chromatography was about five times of commercially agarose chromatography and the specific activity was up to 154 IU/mg protein. Furthermore, we studied the regeneration process of the superporous medium, washing the column with 5 column volumes of 1 mol/L NaOH at a low flow rate, to ensure the chromatographic stability. This purification process is simple, reproducible and suitable for large-scale production.

Puerperal cerebral venous thrombosis (CVT) is a relatively common form of stroke in young women in India. The blood coagulationfactor VII (FVII) R353Q polymorphism increases the risk for venous thrombosis. Our aim was to investigate the association of FVII R353Q polymorphism with the risk of puerperal CVT. A total of 100 women with puerperal CVT and 102 age-matched women without postpartum complications were investigated. FVII R353Q genotypes were identified using restriction fragment length polymorphism analysis. Our results showed that the homozygous FVII 353QQ genotype was present in 9% and 8% of patients and controls, respectively; and 42% of patients and 31.4% of controls had the heterozygous 353RQ genotype (odds ratio = 1.55, 95% confidence interval = 0.89-2.70; p = 0.243). Our findings suggest that the FVII R353Q polymorphism is not associated with increased risk for CVT occurring during the puerperal period in Indian women.

Inhibitors of coagulationfactor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin-activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non-protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

Factor VIII (FVIII) is a key protein in blood coagulation, deficiency or malfunction of which causes Haemophilia A. The sole cure for this condition is intravenous administration of FVIII, whose membrane-bound structure we have studied by Cryo-electron microscopy and image analysis. Self-assembled lipid nanotubes were optimised to bind FVIII at close to native conditions. The tubes diameter was constant at 30 nm and the lipid bilayer resolved. The FVIII molecules were well defined, forming an 8.5 nm thick outer layer, and appeared to reach the hydrophobic core of the bilayer. The two known FVIII atomic models were superimposed with the averaged 2D protein densities. The insertion of the FVIII within the membrane was evaluated, reaffirming that the membrane-binding C2 or C1-C2 domain(s) fully penetrate the outer leaflet of the lipid layer. The presented results lay the basis for new models of the FVIII overall orientation and membrane-binding mechanism.

In the current study, we compared the effects of low- and high-flow anesthesia techniques on hemorheology and coagulation parameters in patients who received sevofluran. Forty patients classified as Risk Group I-II according to American Society of Anesthesiologists' (ASA) guidelines who were scheduled to undergo general anesthesia were randomly assigned to one of two groups. Low-flow anesthesia was administered to the first group, and high-flow anesthesia was used in the second group. Blood samples were obtained in the preoperative and peroperative periods (at 60 and 120 min) for determination of blood and plasma viscosity, plasma oncotic pressure, international normalized ratio (INR), phorotrombin time (PT), activated partial phorotrombin time (aPTT) and fibrinogen. Blood was also drawn for analysis of factor VIII (FVIII) activity, which was measured in the preoperative period and at postoperative six hour. The peroperative plasma viscosity was significantly low in Group 1 relative to Group 2. aPTT was significantly elevated at 60 minutes in Group 1 relative to Group 2, but the increase at 120 minutes was not significant. The effects of low-flow anesthesia on hemorheology were greater than those of high-flow anesthesia.

In the present study, a plant binary expression vector PG-pRD12-hFIX (where PG is polygalacturonase) harbouring the hFIX (human coagulationFactor IX) gene was constructed and introduced into tomato (Lycopersicon esculentum) via Agrobacterium tumefaciens-mediated transformation. After kanamycin selection, 32 putative independent transgenic tomato plants were regenerated. PCR and Southern-blot analyses confirmed the transgenic status of some plants. RT (reverse transcription)-PCR analysis for the expression of the introduced gene (hFIX) demonstrated that the hFIX gene was expressed specifically in fruits of the tomato. Western-blot analysis confirmed the presence of a 56 kDa band specific to hFIX in the transformed tomatoes. ELISA results showed that the expression of hFIX protein reached a maximum of 15.84 ng/g fresh weight in mature fruit. A blood-clotting assay demonstrated the clotting activity of the expressed hFIX protein in transgenic tomato fruits. This is the first report on the expression of hFIX in plants, and our research provides potentially valuable knowledge for further development of the plant-derived therapeutic proteins.

Factor XI (FXI) is the zymogen of FXIa, which cleaves FIX in the intrinsic pathway of coagulation. FXI is known to exist as a dimer and interacts with multiple proteins via its 4 apple domains in the “saucer section” of the enzyme; however, to date, no complex crystal structure has been described. To investigate protein interactions of FXI, a large random peptide library consisting of 106 to 107 peptides was screened for FXI binding, which identified a series of FXI binding motifs containing the signature Asp-Phe-Pro (DFP) tripeptide. Motifs containing this core tripeptide were found in diverse proteins, including the known ligand high-molecular-weight kininogen (HK), as well as the extracellular matrix proteins laminin and collagen V. To define the binding site on FXI, we determined the crystal structure of FXI in complex with the HK-derived peptide NPISDFPDT. This revealed the location of the DFP peptide bound to the FXI apple 2 domain, and central to the interaction, the DFP phenylalanine side-chain inserts into a major hydrophobic pocket in the apple 2 domain and the isoleucine occupies a flanking minor pocket. Two further structures of FXI in complex with the laminin-derived peptide EFPDFP and a DFP peptide from the random screen demonstrated binding in the same pocket, although in a slightly different conformation, thus revealing some flexibility in the molecular interactions of the FXI apple 2 domain. PMID:27006387

The activation and regulation of coagulationFactor XIII (FXIII) protein has been the subject of active research for the past three decades. Although discrete evidence exists on various aspects of FXIII activation and regulation a combinatorial structure/functional view in this regard is lacking. In this study, we present results of a structure/function study of the functional chain of events for FXIII. Our study shows how subtle chronological submolecular changes within calcium binding sites can bring about the detailed transformation of the zymogenic FXIII to its activated form especially in the context of FXIIIA and FXIIIB subunit interactions. We demonstrate what aspects of FXIII are important for the stabilization (first calcium binding site) of its zymogenic form and the possible modes of deactivation (thrombin mediated secondary cleavage) of the activated form. Our study for the first time provides a structural outlook of the FXIIIA2B2 heterotetramer assembly, its association and dissociation. The FXIIIB subunits regulatory role in the overall process has also been elaborated upon. In summary, this study provides detailed structural insight into the mechanisms of FXIII activation and regulation that can be used as a template for the development of future highly specific therapeutic inhibitors targeting FXIII in pathological conditions like thrombosis. PMID:27453290

Generation of coagulationfactor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin- activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin- activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes

Objectives: Inherited thrombophilia might lead to recurrent pregnancy loss (RPL). The aim of the study was to estimate the prevalence of V coagulationfactor polymorphisms related with inherited thrombophilia among women in Malopolska region.Material and methods: Group of 136 women, who experienced at least 2 unexplained, idiopathic pregnancy loss. 106 healthy women having at least one uncomplicated pregnancy and delivered healthy children constituted a control group. Each patient were examined for factor V Leiden (FVL) and mutation 1328 T>C of factor V gene with use of real –time PCR and Taq-Man probes.Results: Among patients with RPL inhabiting region of Malopolska compared to control group occurred higher prevalence of FVL and mutation 1328 T>C. There is coincidence of polymorphism 1328 T>C of factor V gene and FVL in group of early and late RPL.Conclusions: TC genotype of 1328 T>C mutation carriers reveal tendency toward RPL below 7 weeks of pregnancy.Based on results of these findings inherited thrombophilia evaluation in patients after two or more RPL should be recommended.

The liver plays a central role in the clotting process. In this organ are sintetizated the major part of the coagulationfactors. Historically, was considered that alteration in liver function causes important bleeding disorders. However, actual evidence is not in agreement with this asseveration. Decreased synthesis of clotting and inhibitor factors, decrease clearance of activated factors, quantitative and qualitative platelet defects, hyperfibrinolysis and intravascular coagulation are some of the defects observed in liver diseases. Thrombotic events, even if rare in cirrhotic patients, occur manly in the portal and mesenteric veins. The aim of the present work is to review the present evidence in coagulation disorders and liver disease.

Blood coagulationfactor XIII (FXIII) is a protransglutaminase that becomes activated by the concerted action of thrombin and Ca{sup 2+} in the final stage of the clotting cascade. In addition to plasma, FXIII also occurs in platelets, monocytes, and monocyte-derived macrophages. While the plasma factor is a heterotetramer consisting of paired A and B subunits (A{sub 2}B{sub 2}), its cellular counterpart lacks the B subunits and is a homodimer of potentially active A subunits (A{sub 2}). The gene coding for the A and B subunits has been localized to chromosomes 6p24-25 and 1q31-32.1, respectively. The genomic as well as the primary protein structure of both subunits has been established. Plasma FXIII circulates in association with its substrate precursor, fibrinogen. Fibrin(ogen) has an important regulatory role in the activation of plasma FXIII, for instance the proteolytic removal of activation peptide by thrombin, the dissociation of subunits A and B, and the exposure of the originally buried active site on the free A subunits. The end result of this process is the formation of an active transglutaminase, which crosslinks peptide chains through {epsilon}({gamma}-glutamyl)lysyl isopeptide bonds. The protein substrates of activated FXIII include components of the clotting-fibrinolytic system, adhesive and contractile proteins. The main physiological function of plasma FXIII is to cross-link fibrin and protect it from the fibrinolytic enzyme plasmin. The latter effect is achieved mainly by covalently linking {alpha}{sub 2} antiplasmin, the most potent physiological inhibitor of plasmin, to fibrin. Plasma FXIII seems to be involved in wound healing and tissue repair, and it is essential to maintaining pregnancy. Cellular FXIII, if exposed to the surface of the cells, might support or perhaps take over the hemostatic functions of plasma FXIII; however, its intracellular role has remained mostly unexplored. 328 refs., 4 figs.

Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.

Total hip arthroplasty (THA) is an effective treatment for end-stage haemophilic arthropathy, and substitution therapy plays a key role in the success of THA. The aim of this study was to evaluate the efficacy of a modified coagulationfactor substitution regime in THA. Nineteen haemophiliac patients (20 hips) who received primary cementless THA were enrolled. Based on World Federation of Haemophilia (WFH) guideline, a modified coagulationfactor substitution regime was adopted. Blood loss, implant survival rates and complications were reviewed, retrospectively. The mean age at surgery was 29.7 years (15-49 years) and the mean follow-up period was 91 months (43-151 months). Mean total blood loss, external blood loss and hidden blood loss were 3543 (1494-7576), 1435 (600-3440), and 2110 ml (534-4402), respectively. Mean intraoperative blood loss and postoperative drainage were 715 (300-2000) and 713 ml (200-2950), respectively. Mean red blood cell transfusion used was 5 U (0-14). All prostheses were found to have bony ingrowth. One patient had hematoma formation in the thigh and one with a lower limb deep vein thrombosis, postoperatively. Other complications included one skin ulcer, one femur splitting fracture, and one transient neuropraxia. Intraoperative blood loss and wound drainage, in our study, were similar to that in haemophiliac patients and nonhaemophilic patients in literature. This supports the efficacy of the modified coagulationfactor substitution strategy in our study.

Factor VIII and von Willebrand factor are two plasma proteins essential for effective hemostasis. In vivo, they form a non-covalent complex whose association appears to be metal ion dependent. However, a precise definition of the nature of the molecular forces governing their association remains to be defined, as does their binding affinity. In this paper we have determined the dissociation constant and stoichiometry for Factor VIII binding to immobilized von Willebrand factor. The data demonstrate that these proteins interact saturably and with relatively high affinity. Computer assisted analyses of the Scatchard data favour a two site binding model. The higher affinity site was found to have a Kd of 62 (+/- 13) x 10(-12) M while that of the lower affinity site was 380 (+/- 92) x 10(-12) M. The density of Factor VIII binding sites (Bmax) present on von Willebrand factor was 31 (+/- 3) pM for the high affinity binding site and 46 (+/- 6) pM for the lower site, corresponding to a calculated Factor VIII: von Willebrand factor binding ratio of 1:33 and 1:23, respectively.

Tissue factor (TF) is a cell surface receptor for coagulationfactor VII (fVII). The TF-activated fVII (fVIIa) complex is an essential initiator of the extrinsic blood coagulation process. Interactions between cancer cells and immune cells via coagulationfactors and adhesion molecules can promote progression of cancer, including epithelial ovarian cancer (EOC). This process is not necessarily advantageous, as tumor tissues generally undergo hypoxia due to aberrant vasculature, followed by reduced access to plasma components such as coagulationfactors. However, hypoxia can activate TF expression. Expression of fVII, intercellular adhesion molecule-1 (ICAM-1), and multiple pro-inflammatory cytokines can be synergistically induced in EOC cells in response to hypoxia along with serum deprivation. Thus, pro-inflammatory responses associated with the TF-fVIIa–ICAM-1 interaction are expected within hypoxic tissues. Tumor tissue consists of multiple components such as stromal cells, interstitial fluid, albumin, and other micro-factors such as proton and metal ions. These factors, together with metabolism reprogramming in response to hypoxia and followed by functional modification of TF, may contribute to coagulationfactor-driven inflammatory responses in EOC tissues. The aim of this review was to describe potential coagulationfactor-driven inflammatory responses in hypoxic EOC tissues. Arguments were extended to clinical issues targeting this characteristic tumor environment. PMID:28417928

The naturally occurring M358R mutation of the plasma serpin α{sub 1}-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg–Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg–Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10{sup 2} M{sup −1}sec{sup −1}. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. - Highlights: • The inhibitory specificity of the serpin alpha-1-proteinase inhibitor (API) is sharply altered in the M358R variant. • API M358R forms denaturation-resistant complexes with coagulationfactor VIIa at a rate accelerated by tissue factor but unaffected by heparin. • Complex formation was shown by gel-based assays and quantified kinetically by inhibition of FVIIa-dependent amidolysis.

Coagulationfactor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (ki/ka = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis. PMID:25688860

Lipid rafts are an initiation site for many different signals. Recently, we reported that an EGF domain in activated coagulationfactor IX (EGF-F9) increases lipid raft formation and accelerates cell migration. However, the detailed mechanism is not well understood. This study aimed to evaluate the effects of EGF-F9 on the cell membrane. A431 cells (derived from human squamous cell carcinoma) were treated with recombinant EGF-F9. Cells were immunocytochemically stained with probes for lipid rafts or phosphatidylserine (PS). After 3 min of treatment with EGF-F9, cholera toxin subunit B (CTxB) binding domains emerged at the adhesive tips of filopodia. Subsequently, CTxB staining was observed on the filopodial shaft. Finally, large clusters of CTxB domains were observed at the edge of cell bodies. Markers for lipid rafts, such as caveolin-1 and a GPI anchored protein, co-localized with CTxB. Staining with annexin V and XII revealed that PS was exposed at the tips of filopodia, translocated on filopodial shafts, and co-localized with CTxB at the rafts. Immunocytochemistry showed that scramblase-1 protein was present at the filopodial tips. Our data indicates that EGF-F9 accelerates PS exposure around the filopodial adhesion complex and induces clustering of lipid rafts in the cell body. PS exposure is thought to occur on cells undergoing apoptosis. Further study of the function of the EGF-F9 motif in mediating signal transduction is necessary because it is shared by a number of proteins.

Human adenoviruses from multiple species bind to coagulationfactor X (FX), yet the importance of this interaction in adenovirus dissemination is unknown. Upon contact with blood, vectors based on adenovirus serotype 5 (Ad5) binds to FX via the hexon protein with nanomolar affinity, leading to selective uptake of the complex into the liver and spleen. The Ad5:FX complex putatively targets heparan sulfate proteoglycans (HSPGs). The aim of this study was to elucidate the specific requirements for Ad5:FX-mediated cellular uptake in this high-affinity pathway, specifically the HSPG receptor requirements as well as the role of penton base-mediated integrin engagement in subsequent internalisation. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins/heparan sulfates significantly decreased FX-mediated Ad5 cell binding in vitro and ex vivo. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 cellular uptake. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration of adenovirus. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or αv integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface involves HSPG sulfation. Interestingly, Ad5 vectors ablated for αv integrin binding showed markedly delayed cell entry, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and transport following surface binding mediated through FX. This study therefore integrates the established model of αv integrin-dependent adenoviral infection with the high-affinity FX-mediated pathway. This has important implications for mechanisms that define

N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulationfactor VII (FVII) in CHO-K1 and 293A cells. Human FVII has two N-glycosylation sites (N145 and N322). Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans, respectively. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans. Immediately after pulse, most labeled intracellular FVII had one N-glycan, but during a 1-h chase, the vast majority was processed into FVII with two N-glycans, demonstrating posttranslational N-glycosylation of FVII. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322. The posttranslational N-glycosylation appeared to take place in the same time frame as the folding of nascent FVII into a secretion-competent conformation, indicating a link between the two processes. We propose that the cotranslational conformation(s) of FVII are unfavorable for glycosylation at N332, whereas a more favorable conformation is obtained during the posttranslational folding. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins.

Hereditary factor XI deficiency is a rare bleeding disorder with worldwide distribution. In Austrian patients only one mutation leading to congenital factor XI deficiency has been reported. In the present study, we identified the molecular basis of factor XI deficiency in three Austrian patients. Patients attended hospital for other reasons than bleeding disorders. Routine laboratory tests revealed prolonged APTTs due to decreased factor XI levels. We performed automated fluorescent sequencing of the promotor region, exons 1-15 and the flanking intronic regions of the factor XI gene. The mutations found were confirmed by restriction enzyme analysis or sequencing of the non-coding strand. Fluorescent sequencing revealed two novel mutations, the nonsense mutation Gln116X (443C>T) in exon 5 and a deletion of Ile197 and Asp198 (687_692delTCGACA) in exon 7. Furthermore, we detected a heterozygous A>G exchange at the third nucleotide of IVS6 (IVS 6 +3A>G), which had already been reported in a FXI deficient individual of French Basque origin. While the IVS 6 +3A>G decreases the calculated splice consensus score from 0.98 in the wild type to 0.56 in the altered sequence and therefore interferes with the consensus splice sequence, the complete loss of the two amino acids Ile197 and Asp198 is expected to interfere with the steric structure and hence the functions of the third apple domain. The Gln116X leads to a premature termination codon resulting in a lack of the light as well as parts of the heavy chain of the FXI protein, most likely resulting in rapid degradation of the truncated mRNA.

A new type of hereditary angioedema (type III) affecting mainly women with normal C1-inhibitor level and function has been described. Exposition to estrogens is an important precipitating factor. Recently, a missense mutation in the gene of the blood coagulationfactor XII (Hageman factor) has been reported in a few families with this type of hereditary angioedema. To study a patient and her family with recurrent swelling attacks during pregnancy. Complement factors C3 and C4 as well as C1-inhibitor level and function were determined. Genomic DNA was isolated from venous blood samples and screened for mutations in the coagulationfactor XII gene. C3 and C4 levels as well as C1-inhibitor level and function were normal. A missense mutation Thr309Lys was identified in factor XII gene with a heterozygotic pattern. This mutation was also identified in the mother of the patient, her daughter and her son. These results support that the mentioned mutation in factor XII gene causes hereditary angioedema type III.

Coagulationfactor XIII, a zymogen present in blood as a tetramer (A2B2) of A- and B-domains, is one of the components of many ''wound sealants'' which are proposed for use or currently in use as effective hemostatic agents, sealants and tissue adhesives in surgery. After activation by ?-thrombin cleavage, coagulationfactor XIII A-domain, a transglutaminase, is formed and catalyzes the covalent crosslinking of the ?- and ?-chains of linear fibrin to form homopolymers, which can quickly stop bleeding. We have successfully expressed the A-domain of factor XIII in both plant cell cultures and whole plants. Transgenic plant cell culture allows a rapid method for testing production feasibility while expression in whole plants demonstrates an economic production system for recombinant human plasma-based proteins. The expressed factor XIII A-domain had a similar size as that of human plasma-derived factor XIII. Crude plant extract containing recombinant factor XIII A-domain showed transglutaminase activity with monodansylcadaverine and casein as substrates and crosslinking activity in the presence of linear fibrin. The expression of factor XIII A-domain was not affected by plant leaf position.

Hereditary angioedema is characterized by recurrent skin swelling, abdominal pain attacks, and potentially life-threatening upper airway obstruction. The two classic types are both caused by mutations within the complement C1 inhibitor gene. A recently described new type does not show a deficiency of C1 inhibitor and affects almost exclusively women. We screened twenty unrelated index patients with this new type of hereditary angioedema for mutations in the coagulationfactor XII gene. Two different missense mutations were identified in exactly the same position within exon 9 of the F12 gene. 'Mutation 1' (1032C-->A), encountered in five patients, predicts a threonine-to-lysine substitution (Thr309Lys). 'Mutation 2' (1032C-->G), observed in one patient, results in a threonine-to-arginine substitution (Thr309Arg). The predicted structural and functional impact of the mutations, their absence in 145 healthy controls, and their co-segregation with the phenotype in five families provide strong support that they cause disease.

Exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane is thought to play a critical role in tissue factor (TF) decryption. Recent molecular dynamics simulation studies suggested that the TF ectodomain may directly interact with PS. To investigate the potential role of TF direct interaction with the cell surface phospholipids on basal TF activity and the enhanced TF activity following the decryption, one or all of the putative PS-interactive residues in the TF ectodomain were mutated and tested for their coagulant activity in cell systems. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A- exhibited a similar TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A, and TFK166A, was reduced substantially. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% inhibition. Mutation of all nine lipid binding residues together did not further decrease the activity of TF compared to TFG164A. A similar fold increase in TF activity was observed in wild-type TF and all TF mutants following the treatment of THP-1 cells with either calcium ionomycin or HgCl2, two agents that are commonly used to decrypt TF. Overall, our data show that a few select TF residues that are implicated in interacting with PS contribute to the TF coagulant activity at the cell surface. However, our data also indicate that TF regions outside of the putative lipid binding region may also contribute to PS-dependent decryption of TF. PMID:27348126

It is unknown if a lower size limit exists for human blood coagulation under flow over physiologically vessel wall triggers as small as a single collagen fiber. Prior determinations of the smallest sized surface stimuli necessary for clotting of human blood, defined as the patch size threshold, have not deployed whole blood, hemodynamic flow, and platelet adhesive stimuli. For whole blood perfused in microfluidic devices, we report that steady venous flow (wall shear rate, 100 s−1) was sufficient to drive platelet deposition on 20-micron long zones of collagen fibers or on a single fiber. With tissue factor (TF)-coated collagen, flowing blood generated robust platelet deposits, platelet-localized thrombin, and fibrin on a single collagen fiber, thus demonstrating the absence of a physiological patch size threshold under venous flow. In contrast, at arterial wall shear rate (1000 s−1) with TF present, essentially no platelet or fibrin deposition occurred on 20-micron collagen zones or on a single collagen fiber, demonstrating a patch threshold, which was overcome by pre-coating the collagen with von Willebrand factor (VWF). For venous flows, human blood can clot on one of the smallest biological units of a single collagen fiber presenting TF. For arterial flows, VWF together with TF allows human blood to generate thrombin and fibrin on a patch stimulus as limited as a single collagen fiber. VWF-dependent platelet adhesion represents a particle-based sensing mechanism of micron-scale stimuli that then allows amplification of the molecular components of TF-driven thrombin and fibrin production under arterial flow. PMID:27339024

It is unknown if a lower size limit exists for human blood coagulation under flow over physiological vessel wall triggers as small as a single collagen fiber. Prior determinations of the smallest sized surface stimuli necessary for clotting of human blood, defined as the patch size threshold, have not deployed whole blood, hemodynamic flow, and platelet adhesive stimuli. For whole blood perfused in microfluidic devices, we report that steady venous flow (wall shear rate, 100 s(-1)) was sufficient to drive platelet deposition on 20 micron long zones of collagen fibers or on a single fiber. With tissue factor (TF)-coated collagen, flowing blood generated robust platelet deposits, platelet-localized thrombin, and fibrin on a single collagen fiber, thus demonstrating the absence of a physiological patch size threshold under venous flow. In contrast, at arterial wall shear rate (1000 s(-1)) with TF present, essentially no platelet or fibrin deposition occurred on 20 micron collagen zones or on a single collagen fiber, demonstrating a patch threshold, which was overcome by pre-coating the collagen with von Willebrand factor (vWF). For venous flows, human blood can clot on one of the smallest biological units of a single collagen fiber presenting TF. For arterial flows, vWF together with TF allows human blood to generate thrombin and fibrin on a patch stimulus as limited as a single collagen fiber. vWF-dependent platelet adhesion represents a particle-based sensing mechanism of micron-scale stimuli that then allows amplification of the molecular components of TF-driven thrombin and fibrin production under arterial flow.

Arterial thrombosis is the result of a complex and well-orchestrated set of events where interactions between platelets and leukocytes are intertwined with enzymatic reactions of the coagulation system. Here, we present a contemporary panorama of arterial thrombosis and provide a framework the interventionalist can use to understand the current antithrombotic pharmacotherapies and recognize the role of therapies that have yet to be developed. We analyze thrombosis in the context of plaque rupture and vascular injury and describe the interactions between platelets and the subendothelium. We then discuss platelet-leukocyte interactions, emphasizing the inflammatory nature of thrombosis and how this relates to vessel restenosis following angioplasty. The different reactions of the coagulation system are described not from an isolated perspective, but are integrated into the sequence of cell-cell interactions that parallel them. Finally, we describe the mechanisms that terminate the thrombotic response. Copyright 2003 Wiley-Liss, Inc.

The effect of pre-eclampsia on coagulation and fibrinolysis in newborns is still under investigation. We have evaluated several coagulation and fibrinolysis parameters in umbilical cord blood of 20 newborns from pre-eclamptic women and of 40 newborns from normotensive women with similar gestational age. Additionally, the presence of factor V Leiden and prothrombin G20210A mutation in cord blood has been assessed. Neonates from pre-eclamptic women exhibited significantly lower birth weight (2.48 +/- 0.92 versus 2.88 +/- 0.68 kg, P < 0.05) and were more frequently admitted to the neonatal intensive care unit (45 versus 20%, P < 0.01) as compared with neonates from normotensive women. Cord blood protein C antigen and activated protein C resistance mean levels were slightly higher in the group of neonates from pre-eclamptic mothers. Fibrinogen levels were lower in this group as compared with control newborns (132.17 +/- 46.97 versus 156.08 +/- 49.58 mg%, P < 0.02), and unrelated to birth weight. No significant differences between cases and controls were found in plasminogen activator inhibitor-1 or tissue plasminogen activator cord blood levels. Heterozygous prothrombin 20210A was found in three newborns from normotensive mothers, whereas no factor V Leiden mutation was found in either group. In conclusion, pre-eclampsia seems to have only mild effects on coagulation and fibrinolytic factors in the cord blood of newborns. Since no excess of common polymorphisms predisposing to thrombosis was found in newborns from pre-eclamptic mothers, it is unlikely that the carriership status of these genetic defects of newborns influences the adverse pregnancy/neonatal outcomes.

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulationfactors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulationfactors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulationfactor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulationfactors.

Dermatitis herpetiformis (DH) and bullous pemphigoid (BP) are skin diseases associated with eosinophilic and neutrophilic infiltrations. Although chemokines are critical for the selective accumulation and activation of various leukocyte subsets in the inflammatory process, there are few findings concerning inflammatory cells and production of coagulationfactors in blistering diseases. Skin biopsies were taken from 14 patients with DH, 27 with BP, and 20 control subjects. The localization and expression of tissue factor (TF) in skin lesions and perilesional skin were studied by immunohistochemistry and confirmed by Western Blot. Moreover the plasma concentrations of TF were measured by immunoassays. D dimers, fibrinogen, and selected coagulation parameters were measured by routine methods. Expression of TF in the epidermis and in inflammatory influxed cells in dermis was detected in skin biopsies from BP patients. Examined TF expression was detected in perilesional skin of all BP patients too. The expression of TF was not observed in biopsies from healthy people and DH patients. The findings of the study show an increased expression of tissue factor in the lesional and perilesional skin of patients with bullous pemphigoid. The difference in chemokine pattern expression and variations in the cellular infiltration in BP and DH cause variable expression of TF. PMID:27057091

Blood coagulation occurs through a cascade of enzymes and cofactors that produces a fibrin clot, while otherwise maintaining hemostasis. The 11 human coagulationfactors (FG, FII-FXIII) have been identified across all vertebrates, suggesting that they emerged with the first vertebrates around 500 Ma. Human FVIII, FIX, and FXI are associated with thousands of disease-causing mutations. Here, we evaluated the strength of selective pressures on the 14 genes coding for the 11 factors during vertebrate evolution, and compared these with human mutations in FVIII, FIX, and FXI. Positive selection was identified for fibrinogen (FG), FIII, FVIII, FIX, and FX in the mammalian Primates and Laurasiatheria and the Sauropsida (reptiles and birds). This showed that the coagulation system in vertebrates was under strong selective pressures, perhaps to adapt against blood-invading pathogens. The comparison of these results with disease-causing mutations reported in FVIII, FIX, and FXI showed that the number of disease-causing mutations, and the probability of positive selection were inversely related to each other. It was concluded that when a site was under positive selection, it was less likely to be associated with disease-causing mutations. In contrast, sites under negative selection were more likely to be associated with disease-causing mutations and be destabilizing. A residue-by-residue comparison of the FVIII, FIX, and FXI sequence alignments confirmed this. This improved understanding of evolutionary changes in FVIII, FIX, and FXI provided greater insight into disease-causing mutations, and better assessments of the codon sites that may be mutated in applications of gene therapy.

Blood coagulation occurs through a cascade of enzymes and cofactors that produces a fibrin clot, while otherwise maintaining hemostasis. The 11 human coagulationfactors (FG, FII–FXIII) have been identified across all vertebrates, suggesting that they emerged with the first vertebrates around 500 Ma. Human FVIII, FIX, and FXI are associated with thousands of disease-causing mutations. Here, we evaluated the strength of selective pressures on the 14 genes coding for the 11 factors during vertebrate evolution, and compared these with human mutations in FVIII, FIX, and FXI. Positive selection was identified for fibrinogen (FG), FIII, FVIII, FIX, and FX in the mammalian Primates and Laurasiatheria and the Sauropsida (reptiles and birds). This showed that the coagulation system in vertebrates was under strong selective pressures, perhaps to adapt against blood-invading pathogens. The comparison of these results with disease-causing mutations reported in FVIII, FIX, and FXI showed that the number of disease-causing mutations, and the probability of positive selection were inversely related to each other. It was concluded that when a site was under positive selection, it was less likely to be associated with disease-causing mutations. In contrast, sites under negative selection were more likely to be associated with disease-causing mutations and be destabilizing. A residue-by-residue comparison of the FVIII, FIX, and FXI sequence alignments confirmed this. This improved understanding of evolutionary changes in FVIII, FIX, and FXI provided greater insight into disease-causing mutations, and better assessments of the codon sites that may be mutated in applications of gene therapy. PMID:25158795

Monoclonal antibodies to factor VIII antigen (VIII:Ag) and von Willebrand factor (vWf:Ag) were immobilised on Sephacryl S-1000 and tested for their ability to deplete normal human citrated plasma of factor VIII. A combination of two antibodies to VIII:Ag and one antibody to vWf:Ag was required to produce plasma containing less than 0.01 IU/ml. Its performance in the one stage coagulation assay of VIII:C was equivalent to that of congenital VIII deficient plasma for the assay of normal and haemophilic plasma and factor VIII concentrates. Storage of freeze dried aliquots of this product at -20 degrees C, +4 degrees C, and 37 degrees C showed that it could be used as a substrate for at least six months when stored at temperatures +4 degrees C and below. PMID:3133400

Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. The authors identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate for factor X in the presence of factor VIII, phospholipids, and Ca{sup 2+}, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311 {yields} Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

Coagulationfactor II G20210A and coagulationfactor V (Leiden) G1691A single nucleotide polymorphisms (SNPs) are major inherited risk factors of venous thromboembolism. In view of the heterogeneity in their world distribution and lack of sufficient information about their distribution among Chechans, we addressed the prevalence of these SNPs in the Chechan population in Jordan, a genetically isolated population. Factor II G20210A and factor V Leiden SNPs were analysed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method and Amplification refractory mutation detection system (ARMS) respectively in 120 random unrelated subjects from the Chechan population in Jordan. Among the subjects studied for factor II G20210A mutation there were three individuals carrying this mutation as heterozygous (one female and two male), giving a prevalence of 2.5 % and an allele frequency of 1.25 %. No homozygous factor II allele was found. Factor V Leiden G1691A mutation was detected as heterozygous in 22 of 120 of individuals (17 female and five male) indicating a prevalence of 18.3 % and allele frequency of 9.2 %. No homozygous allele was found. Our results indicated that prevalence of factor II G20210A mutation in the Chechan population is similar to prevalence in Jordan and Caucasian populations (1-6 %) while the prevalence of factor V Leiden was higher in the Chechan population compared to Jordan and Caucasian populations (2-15 %).

Objective Coagulationfactor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was aimed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo. Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal thrombus formation without affecting proximal platelet adhesion. PMID:26769048

Recombinant coagulationfactor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulationfactor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine β-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

Severe sepsis is almost invariably associated with systemic activation of coagulation. There is ample evidence that demonstrates a wide-ranging cross-talk between hemostasis and inflammation, which is probably implicated in the pathogenesis of organ dysfunction in patients with sepsis. Inflammation not only leads to initiation and propagation of coagulation activity, but coagulation also markedly influences inflammation. Molecular mechanisms that play a role in inflammation-induced effects on coagulation have been recognized in much detail. Pro-inflammatory cells and cyto- and chemokines can activate the coagulation system and downregulate crucial physiological anticoagulant mechanisms. Initiation of coagulation activation and consequent thrombin generation is caused by expression of tissue factor on activated monocytes and endothelial cells and is ineffectually offset by tissue factor pathway inhibitor. At the same time, endothelial-associated anticoagulant pathways, in particular the protein C system, is impaired by pro-inflammatory cytokines. Also, fibrin removal is severely obstructed by inactivation of the endogenous fibrinolytic system, mainly as a result of upregulation of its principal inhibitor, plasminogen activator inhibitor type 1 (PAI-1). Increased fibrin generation and impaired break down lead to deposition of (micro)vascular clots, which may contribute to tissue ischemia and ensuing organ dysfunction. The foundation of the management of coagulation in sepsis is the explicit and thorough treatment of the underlying disorder by antibiotic treatment and source control measures. Adjunctive strategies focused at the impairment of coagulation, including anticoagulants and restoration of physiological anticoagulant mechanisms, may supposedly be indicated and have been found advantageous in experimental and initial clinical trials.

Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.

Organic polymers are commonly used as coagulants or flocculants in pretreatment for microfiltration (MF). These high molecular weight compounds are potential membrane foulants when carried over to the MF filters. This study examined fouling of three MF membranes of different materials by three commonly used water treatment polymers: poly(diallyldimethylammonium) chloride (pDADMAC), polyacrylamide (PAM), and poly(acrylic acid-co-acrylamide (PACA) with a wide range of molecular weights. The effects of polymer molecular characteristics, membrane surface properties, solution condition and polymer concentration on membrane fouling were investigated. Results showed severe fouling of microfiltration membranes at very low polymer concentrations, suggesting that residual polymers carried over from the coagulation/flocculation basin can contribute significantly to membrane fouling. The interactions between polymers and membranes depended strongly on the molecular size and charge of the polymer. High molecular weight, positively charged polymers caused the greatest fouling. Blockage of membrane pore openings was identified as the main fouling mechanism with no detectable internal fouling in spite of the small molecular size of the polymers relative to the membrane pore size. Solution conditions (e.g., pH and calcium concentration) that led to larger polymer molecular or aggregate sizes resulted in greater fouling.

Rotational thromboelastometry (ROTEM) and thromboelastography (TEG) have been increasingly used to diagnose acute coagulopathy and guide blood transfusion. The tests are routinely performed using different triggering activators such as tissue factor and kaolin, which activate different pathways yielding different results. To optimize the global blood coagulation assays using ROTEM and TEG, we conducted a comparative study on the activation methods employing tissue factor and kaolin at different concentrations as well as standard reagents as recommended by the manufacturer of each device. Key parameter values were obtained at various assay conditions to evaluate and compare coagulation and fibrinolysis profiles of citrated whole blood collected from healthy volunteers. It was found that tissue factor reduced ROTEM clotting time and TEG R, and increased ROTEM clot formation time and TEG K in a concentration-dependent manner. In addition, tissue factor affected ROTEM alpha angle, and maximum clot firmness, especially in the absence of kaolin activation, whereas both ROTEM and TEG clot lysis (LI30, CL30, and LY30) remained unaffected. Moreover, kaolin reduced ROTEM clotting time and TEG R and K, but to a lesser extent than tissue factor, in-tem and ex-tem. Correlations in all corresponding parameters between ROTEM and TEG were observed, when the same activators were used in the assays compared with lesser correlations between standard kaolin TEG and ROTEM (INTEM/EXTEM). The two types of viscoelastic point-of-care devices provide different results, depending on the triggering reagent used to perform the assay. Optimal assay condition was obtained to reduce assay time and improve assay accuracy.

Traditional biochemistry of contact activation of blood coagulation suggesting that anionic hydrophilic surfaces are specific activators of the cascade is inconsistent with known trends in protein adsorption. To investigate contact activation reactions, a chromogenic assay was used to measure prekallkrein (PK) hydrolysis to kallikrein (Kal) by activated factor XII (FXIIa) at test hydrophilic (clean glass) and hydrophobic (silanized glass) surfaces in the presence of bovine serum albumin (BSA). Hydrolysis of PK by FXIIa is detected after contact of the zymogen FXII with a test hydrophobic surface only if putatively-adsorbed FXIIa is competitively displaced by BSA. By contrast, FXIIa activity is detected spontaneously following FXII activation by a hydrophilic surface and requires no adsorption displacement. These results (i) show that an anionic hydrophilic surface is not a necessary cofactor for FXIIa-mediated hydrolysis of PK, (ii) indicate that PK hydrolysis does not need to occur by an activation complex assembled directly on an anionic, activating surface, (iii) confirms that contact activation of FXII (autoactivation) is not specific to anionic hydrophilic surfaces, and (iv) demonstrates that protein-adsorption competition is an essential feature that must be included in any comprehensive mechanism of surface-induced blood coagulation.

Coagulationfactor XII (FXII) is a key protein in the intrinsic coagulation and kallikrein–kinin pathways. It has been found that negative surfaces and amyloids, such as Aβ fibrils, can activate FXII. Additionally, it has been suggested that FXII simulates cells and that it plays an important role in thrombosis. To date, no structural data on FXII have been deposited, which makes it difficult to support any hypothesis on the mechanism of FXII function. The crystal structure of the FnI-EGF-like tandem domain of FXII presented here was solved using experimental phases. To determine the phases, a SIRAS approach was used with a native and a holmium chloride-soaked data set. The holmium cluster was coordinated by the C-terminal tails of two symmetry-related molecules. Another observation was that the FnI domain was much more ordered than the EGF-like domain owing to crystal packing. Furthermore, the structure shows the same domain orientation as the homologous FnI-EGF-like tandem domain of tPA. The plausibility of several proposed inter­actions of these domains of FXII is discussed. Based on this FXII FnI-EGF-like structure, it could be possible that FXII binding to amyloid and negatively charged surfaces is mediated via this part of FXII. PMID:23385745

Coagulation is a dynamic process and the understanding of the blood coagulation system has evolved over the recent years in anaesthetic practice. Although the traditional classification of the coagulation system into extrinsic and intrinsic pathway is still valid, the newer insights into coagulation provide more authentic description of the same. Normal coagulation pathway represents a balance between the pro coagulant pathway that is responsible for clot formation and the mechanisms that inhibit the same beyond the injury site. Imbalance of the coagulation system may occur in the perioperative period or during critical illness, which may be secondary to numerous factors leading to a tendency of either thrombosis or bleeding. A systematic search of literature on PubMed with MeSH terms ‘coagulation system, haemostasis and anaesthesia revealed twenty eight related clinical trials and review articles in last 10 years. Since the balance of the coagulation system may tilt towards bleeding and thrombosis in many situations, it is mandatory for the clinicians to understand physiologic basis of haemostasis in order to diagnose and manage the abnormalities of the coagulation process and to interpret the diagnostic tests done for the same. PMID:25535411

Introduction Vascular malformation is associated with coagulopathies, especially when hemostasis is challenged. Case presentation We present the case of an 11-year-old Hispanic girl with Klippel-Trenaunay-Weber syndrome that developed disseminated intravascular coagulation after minor surgery, which was controlled by blood product transfusions and enoxaparin to address an ongoing consumptive coagulopathy. The patient, however, developed bacteremia and liver trauma that resulted in severe bleeding. To the best of our knowledge, we report here the first known instance of administering recombinant coagulationfactor VIIa to control acute bleeding in a patient with Klippel-Trenaunay-Weber syndrome. Conclusions This case illustrates the concept of enoxaparin maintenance to suppress an ongoing consumptive coagulopathy and the use of recombinant coagulationfactor VIIa to control its potentially fatal severe bleeding episodes. PMID:20302608

The biochemical mechanisms by which activated platelets participate in exposing receptors for the assembly of enzyme-cofactor-substrate complexes at all stages of the blood coagulation cascade are reviewed. Information derived from studies conducted during the last 30 years supports the concept that the initiation of blood coagulation is triggered by exposure of tissue factor at injury sites, leading to the generation of minute quantities of thrombin (limited by tissue factor pathway inhibitor), sufficient to activate platelets, factors XI, VIII, and V, and trigger the consolidation pathway (i.e., the sequential activation of factors XI, IX, X, and prothrombin on the activated platelet surface), leading to the generation of sufficient thrombin to convert fibrinogen to fibrin and effect hemostasis. Platelets localize coagulation to the hemostatic thrombus and protect coagulation enzymes from inhibition by both plasma and platelet inhibitors (e.g., protease nexin 2), thus preventing disseminated intravascular coagulation.

A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulationfactors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).

1. Subcutaneous injections of benzol in rabbits produce marked destructive changes in the hematopoietic organs, especially in the myeloid tissue. 2. Benzol poisoning registers a change not only in the formed elements of the blood, but also in the factors of coagulation. 3. The circulating prothrombin is considerably reduced in amount and in most instances animals in which such a diminution occurs show aplasia of the bone marrow. 4. The association of extreme aplasia of the marrow without a fatal diminution in the circulating prothrombin suggests one of two possibilities: either other tissues and organs in addition to the bone marrow are concerned with prothrombin formation; or a minimum amount of myeloid tissue suffices to maintain the quantity of prothrombin above a dangerous level. PMID:19867880

Because the venous thromboembolisms (VTEs) due to the coagulationfactor V R506Q (FV Leiden) mutation is often seen in Caucasians, the VTE onset in Japan has not been reported. A 34-year-old man from north Africa experiencing sudden dyspnea went to a hospital for advice. The patient had pain in his right leg and a high plasma D-dimer level. A contrast-enhanced computed tomography scan revealed a contrast deficit in the bilateral pulmonary artery and in the right lower extremity. The patient was diagnosed with VTE, and anticoagulation therapy was initiated. Our targeted gene panel sequencing revealed that the occurrence of VTE was attributed to a presence of the FV Leiden mutation. This is the first report demonstrating VTE caused by the FV Leiden mutation in Japan.

Background Despite trauma-induced hypothermic coagulopathy being familiar in the clinical setting, empirical experimentation concerning this phenomenon is lacking. In this study we investigated the effects of hypothermia on thrombin generation, clot formation and global hemostatic functions in an in vitro environment using a whole blood model and thromboelastography (TEG) which can recapitulate hypothermia. Methods Blood was collected from healthy individuals through venipuncture and treated with corn trypsin inhibitor, to block the contact pathway. Coagulation was initiated with 5pM tissue factor at temperatures 37°C, 32°C, and 27°C. Reactions were quenched over time with soluble and insoluble components of each time point analyzed for thrombin generation, fibrinogen consumption, factor (f)XIII activation and fibrin deposition. Global coagulation potential was evaluated through TEG. Results Data showed that thrombin generation in samples at 37°C and 32°C had comparable rates while 27°C had a much lower rate (39.2 ± 1.1 and 43 ± 2.4 nM/min vs 28.6 ± 4.4 nM/min, respectively). Fibrinogen consumption and fXIII activation were highest at 37°C followed by 32°C and 27°C (13.8 ± 2.9 percent/min vs 7.8 ± 1.8 percent/min, respectively). Fibrin formation as seen through clot weights also followed this trend. TEG data showed clot formation was fastest in samples at 37°C and lowest at 27°C. Maximum clot strength was similar for each temperature. Also, percent lysis of clots was highest at 37°C followed by 32°C and then 27°C. Conclusions Induced hypothermic conditions directly affect the rate of thrombin generation and clot formation while global clot stability remains intact. PMID:24331944

A specific blood coagulationfactor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

Two frequently used restriction-enzyme polymorphisms (RFLPs) of coagulant F.IX, TaqI and XmnI, have been examined in five ethnic groups: white Americans, black Americans, East Indians, Chinese, and Malays. There is a distinct "cline" in the frequencies of both polymorphisms, from white Americans to Malays. The rarer type 2 alleles of both polymorphisms, in which middle recognition sites are present--and which in our sample reach their highest frequencies in white Americans--are marginally higher in four groups of Europeans previously reported by others. The frequencies of the rarer alleles are significantly higher in Europeans than in black Americans and East Indians, and these alleles are essentially absent in Chinese and Malays. The frequency of heterozygosity diminishes in the same order, being zero in Malays for both polymorphisms. The polymorphisms are in strong linkage disequilibrium, and in all groups the type 1 allele for TaqI is disproportionately accompanied by the type 1 allele for XmnI. The paucity of type 2 alleles and the low rate of heterozygosity in four non-European groups suggest that the polymorphisms will be of little diagnostic value south of Gibraltar and east of Suez. This prediction is confirmed by the observed haplotype frequencies in the black American and the Oriental groups. PMID:2884869

Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.

Background The aim of this study was to assess whether the quantities of some coagulationfactors in fresh-frozen plasma (FFP) produced from whole blood stored at 4°C for 24 h are adequate for their intended purpose. Materials and methods The amounts of some coagulationfactors (fibrinogen, FV, FVII, FVIII, FX and FXI) in FFP separated from whole blood after storage at 4°C for 24 h were compared with the amounts of the corresponding coagulationfactors in FFP separated from whole blood within 8 h of donation. Results In 98% of the FFP units prepared after 24 h of storage, the levels of fibrinogen, FV, FVII, FX and FXI were greater than 0.5 IU/mL. The concentration of FVIII in the 24 h plasma units was 82% of that found in the FFP units prepared within 8 h of blood collection. In FFP, FVIII, FVII and FX were reduced by 38%, 8% and 3%, respectively, but FV, FXI and fibrinogen were not reduced. Conclusion These data suggest that there is good retention of coagulationfactor activity in plasma produced from whole blood stored at 4ºC for 24 h and that such plasma would be an acceptable product for most patients requiring FFP. PMID:19290079

The aim of this study was to assess whether the quantities of some coagulationfactors in fresh-frozen plasma (FFP) produced from whole blood stored at 4 degrees C for 24 h are adequate for their intended purpose. The amounts of some coagulationfactors (fibrinogen, FV, FVII, FVIII, FX and FXI) in FFP separated from whole blood after storage at 4 degrees C for 24 h were compared with the amounts of the corresponding coagulationfactors in FFP separated from whole blood within 8 h of donation. In 98% of the FFP units prepared after 24 h of storage, the levels of fibrinogen, FV, FVII, FX and FXI were greater than 0.5 IU/mL. The concentration of FVIII in the 24 h plasma units was 82% of that found in the FFP units prepared within 8 h of blood collection. In FFP, FVIII, FVII and FX were reduced by 38%, 8% and 3%, respectively, but FV, FXI and fibrinogen were not reduced. These data suggest that there is good retention of coagulationfactor activity in plasma produced from whole blood stored at 4 masculineC for 24 h and that such plasma would be an acceptable product for most patients requiring FFP.

EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7, a causative agent of diarrhea-associated Hemolytic Uremic Syndrome (D+HUS). The mechanism by which EHEC induces D+HUS has not been fully elucidated. We investigated the effects of EspP on clot formation and lysis in human blood. Human whole blood and plasma were incubated with EspP(WT )at various concentrations and sampled at various time points. Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT), coagulationfactor activities, and thrombelastgraphy (TEG) were measured. Human whole blood or plasma incubated with EspP(WT) was found to have prolonged PT, aPTT, and TT. Furthermore, human whole blood or plasma incubated with EspP(WT) had reduced activities of coagulationfactors V, VII, VIII, and XII, as well as prothrombin. EspP did not alter the activities of coagulationfactors IX, X, or XI. When analyzed by whole blood TEG, EspP decreased the maximum amplitude of the clot, and increased the clot lysis. Our results indicate that EspP alters hemostasis in vitro by decreasing the activities of coagulationfactors V, VII, VIII, and XII, and of prothrombin, by reducing the clot strength and accelerating fibrinolysis, and provide further evidence of a functional role for this protease in the virulence of EHEC and the development of D+HUS.

Background Human blood coagulationfactor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway. Results In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein. Conclusions MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of

Activated platelets facilitate blood coagulation by providing factor V and a procoagulant surface for prothrombinase. Here, we investigated the potential synergy of a potent factor Xa/prothrombinase inhibitor, TAK-442, plus aspirin or clopidogrel in preventing arterial thrombosis and whole blood coagulation. Thrombus formation was initiated by FeCl(3)-induced rat carotid injury. Bleeding time was evaluated with the rat tail transection model. Whole blood coagulation was assessed by thromboelastographic examination (TEG) for which blood obtained from control, aspirin-, or clopidogrel-treated rats was transferred to a TEG analyzer containing, collagen or adenosine diphosphate (ADP), and TAK-442 or vehicle. TAK-442 (3mg/kg, po), aspirin (100mg/kg, po) or clopidogrel (3mg/kg, po) alone had no significant effect on thrombus formation, whereas the combination of TAK-442 with aspirin and clopidogrel remarkably prolonged the time to thrombus formation without additional significant prolongation of bleeding time. TEG demonstrated that the onset of collagen-induced blood coagulation were slightly longer in aspirin-treated rats than control; however, when the blood from aspirin-treated rats was subsequently treated in vitro with 100 nM TAK-442, the onset of clotting was significantly prolonged. In contrast, only marginal prolongation was observed with TAK-442 treatment of blood from control animals. The onset time of ADP-induced blood coagulation was slightly longer in clopidogrel-treated rats compared with control, and it was further extended by TAK-442 treatment. These results demonstrate that blood coagulation can be markedly delayed by the addition of TAK-442 to antiplatelets treatment which could contribute to synergistic antithrombotic efficacy in these settings. (c) 2010 Elsevier Ltd. All rights reserved.

A distinct protein specifically recognized by its strong interaction with Hevea latex lectin (HLL) was detected in the aqueous C-serum fraction of centrifuged fresh latex. This C-serum lectin binding protein (CS-HLLBP) exhibited strong inhibition of HLL-induced hemagglutination. The CS-HLLBP was purified to homogeneity by a protocol that included ammonium sulfate fractionation, size exclusion and ion exchange chromatography. The purified CS-HLLBP had a specific HI titer of 0.23microg ml(-1). Its M(r)s analyzed by SDS-PAGE was ca. 40kDa and that by gel filtration was ca. 204kDa. It has a pI value of 4.7, an optimum activity between pH 6 and10 and was heat stable up to 50 degrees C. The HI activity of CS-HLLBP was abolished upon treatment with chitinase. The CS-HLLBP inhibited HLL-induced rubber particle aggregation in a dose dependent manner. A highly positive correlation between CS-HLLBP activity and rubber yield per tapping was found. The correlations for fresh latex (r=0.98, P<0.01) and dry rubber (r=0.95, P<0.01) were both highly significant. This indicated that the CS-HLLBP might be used as a reliable marker for the mass screening of young seedlings to identify and select clones with potential to be superior producers of rubber. A latex anti-coagulating role of the CS-HLLBP is proposed. The findings described in this 3 paper series have been used to propose a new model of rubber latex coagulation that logically describes roles for the newly characterized latex lectin and the two lectin binding proteins.

When blood coagulation takes place in the presence of calcium ions, alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked to fibrin by activated coagulationFactor XIII (XIIIa) and thereby contributes to the resistance of fibrin to fibrinolysis. It was previously shown that the cross-linking reaction is a reversible one, since the alpha 2PI-fibrinogen cross-linked complex could be dissociated. In the present study we have shown that the alpha 2PI-fibrin cross-linking reaction is also a reversible reaction and alpha 2PI which had been cross-linked to fibrin can be released from fibrin by disrupting the equilibrium, resulting in a decrease of its resistance to fibrinolysis. When the fibrin clot formed from normal plasma in the presence of calcium ions was suspended in alpha 2PI-deficient plasma of buffered saline, alpha 2PI was gradually released from fibrin on incubation. When alpha 2PI was present in the suspending milieu, the release was decreased inversely to the concentrations of alpha 2PI in the suspending milieu. The release was accelerated by supplementing XIIIa or the presence of a high concentration of the NH2-terminal 12-residue peptide of alpha 2PI (N-peptide) which is cross-linked to fibrin in exchange for the release of alpha 2PI. When the release of alpha 2PI from fibrin was accelerated by XIIIa or N-peptide, the fibrin became less resistant to the fibrinolytic process, resulting in an acceleration of fibrinolysis which was proportional to the degree of the release of alpha 2PI. These results suggest the possibility that alpha 2PI could be released from fibrin in vivo by disrupting the equilibrium of the alpha 2PI-fibrin cross-linking reaction, and that the release would result in accelerated thrombolysis. Images PMID:2419360

We studied the ligation of coagulationfactor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.

When surfactant-stabilized biodegradable poly(lactic acid) (PLA) particles are injected into rats, the rate of clearance from blood is fast. The rate can be strongly reduced by using particles made from diblock copolymers of PLA and poly(ethylene oxide) (PLA-PEO), resulting in an increased duration of contact with the components of the coagulation system. Thus, possible adverse effects such as activation of the coagulation cascade could occur. In this paper, the interactions of surfactant-stabilized PLA and PLA-PEO nanoparticle suspensions with the plasma factors of the coagulation system are presented. PLA suspensions stabilized by sodium cholate (PLA-Ch) interact with thrombin, factor V and calcium ions. Formation of complexes and aggregates is induced by addition of calcium ions to PLA-Ch suspensions in the presence or in the absence of plasma. On the contrary, PLA-PEO suspensions are remarkably inert towards the coagulationfactors and calcium ions, even when cholate is present. Steric repulsion owing to the high surface density of PEO is sufficient to avoid strong interations with the proteins and formation of aggregates between particles.

Young healthy volunteers were treated with physical hyperthermia (baths) in order to investigate changes in blood coagulation. Such therapy is used in the treatment of rheumatic diseases. Single hot baths (mean body temperature 38,2-39,9 degrees C) resulted in a rise of fibrinogen, factors IX and XII, maximal amplitude of the thrombelastogram and hemoglobin and in a decrease of plasminogen. In a series of hypothermic baths no additional changes of coagulation or fibrinolysis could be found. The results suggest that hyperthermia causes a tendency to thrombosis.

I. ABSTRACT Malaria remains a highly prevalent disease in more than 90 countries and accounts for at least 1 million deaths every year. Plasmodium falciparum infection is often associated with a procoagulant tonus characterized by thrombocytopenia and activation of the coagulation cascade and fibrinolytic system; however, bleeding and hemorrhage are uncommon events, suggesting that a compensated state of blood coagulation activation occurs in malaria. This article i) reviews the literature related to blood coagulation and malaria in a historic perspective, ii) describes basic mechanisms of coagulation, anticoagulation, and fibrinolysis, iii) explains the laboratory changes in acute and compensated disseminated intravascular coagulation (DIC), iv) discusses the implications of tissue factor (TF) expression in the endothelium of P. falciparum-infected patients, and v) emphasizes the pro-coagulant role of parasitized erythrocytes (pRBC) and activated platelets in the pathogenesis of malaria. This article also presents the ‘Tissue Factor Model’ (TFM) for malaria pathogenesis, which places TF as the interface between sequestration, endothelial cell activation, blood coagulation disorder and inflammation often associated with the disease. The relevance of the coagulation-inflammation cycle for the multiorgan dysfunction and coma is discussed in the context of malaria pathogenesis. PMID:18260002

Clopidogrel, an irreversible ADP-receptor antagonist, inhibits platelet aggregation mediated by reduced activation of glycoprotein receptor IIb/IIIa. Clopidogrel in combination with aspirin has been shown to be superior to aspirin alone for treating unstable angina, but clopidogrel recipients have shown increases in blood loss, transfusion requirements, and rate of reoperation after cardiac surgery. We describe a patient who had taken clopidogrel 75 mg daily until the day prior to coronary artery bypass graft surgery. Severe postoperative bleeding developed and was refractory to conventional hemostatic therapy consisting of 19 units of packed red blood cell concentrates, 16 of fresh frozen plasma, 8 of platelet apheresis concentrates plus high-dose treatment with aprotinin (500.000 kallikrein-inhibiting units/h) and administration of 0.3 microg/kg 1-deamino-8-D-arginine vasopressin (DDAVP). Two reoperations were performed, but surgical hemostasis was not achieved, so 100 microg/kg recombinant activated factor VII was applied to generate sufficient thrombin to stop the bleeding. This treatment approach reduced the bleeding. Then, to promote clot formation and firmness, 2 g of fibrinogen and 1250 IU of factor XIII were administered, and the bleeding finally stopped. No further transfusions were required, and the patient was discharged from the hospital on day 10 after the operation. This case suggests that in clopidogrel-related bleeding refractory to conventional hemostatic therapy, hemostasis may be achieved by a stepwise administration of coagulationfactor concentrates.

Both the complement system and tissue factor (TF), a key initiating component of coagulation, are activated in sepsis, and cross-talk occurs between the complement and coagulation systems. C1-inhibitor (C1-INH) can act as a regulator in both systems. Our aim in this study was to examine this cross-talk by investigating the effects of C1-INH on Escherichia coli-induced haemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultrapurified E. coli lipopolysaccharide (LPS) in the absence or presence of C1-INH or protease-inactivated C1-INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using reverse transcription–quantitative polymerase chain reaction (RT–qPCR), and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1·2 (PTF1·2) and long pentraxin 3 (PTX3) were measured by enzyme-linked immunosorbent assay (ELISA). Cytokines were analysed using a multiplex kit. C1-INH (1·25–5 mg/ml) reduced both LPS- and E. coli-induced coagulation, measured as a reduction of PTF1·2 in plasma, efficiently and dose-dependently (P < 0·05). Both LPS and E. coli induced marked up-regulation of TF mRNA levels and surface expression on whole blood monocytes. This up-regulation was reduced efficiently by treatment with C1-INH (P < 0·05). C1-INH reduced the release of PTX3 (P < 0·05) and virtually all cytokines measured (P < 0·05). Complement activation was inhibited more efficiently with compstatin than with C1-INH. C1-INH inhibited most of the other readouts more efficiently, consistent with additional non-complement-dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1-INH is a broad-spectrum attenuator of the inflammatory and haemostatic responses. PMID:23607270

Establishment of reference intervals for coagulation molecules is important but is costly and sometimes not feasible. Since reference intervals from manufacturers or the literature are mostly out of date or involved Western populations, the authors determined reference intervals for VWF: Ag and eight factors in a Korean population. VWF: Ag, factor VIII (FVIII), FII, FV, FVII, FIX, FX, FXI, and FXII were determined in Korean individuals visiting for routine checkup following the CLSI (Clinical and Laboratory Standards Institute) guidelines. Reagents by Diagnostica Stago were used on the STA Compact Analyzer (Diagnostica Stago). Exclusion criteria were medical history or laboratory findings that could affect the factor levels. Influence of demographic factors was analyzed. Mean +/- 2 x SD or central 95 percentile was used, as appropriate. We obtained data from 266 adults for VWF: Ag, 371 adults for FVIII, and minimum 136 adults for the rest. Reference interval for VWF was 51-176% (52-155% in blood group O and 71-186% for non-O). Reference interval for FVIII was 64-197% (55-150% in O and 77-205% in non-O). Reference interval for FII was 77-121%, FV 81-160%, FVII 68-149%, FIX 67-154%, FX 69-126%, FXI 59-138%, and FXII 48-177%. The medians of VWF: Ag, FVIII, and FIX were significantly higher in the elderly group (> or =60 years). We established local reference intervals for VWF: Ag and eight coagulationfactors in a Korean population according to the CLSI guidelines. Significantly, different reference intervals were obtained in blood group O vs. non-O for VWF: Ag and FVIII. The reference intervals obtained in this study could be adopted in other clinical laboratories after appropriate validation.

Recent studies indicate that the plasma contact system plays an important role in thrombosis, despite being dispensable for hemostasis. For example, mice deficient in coagulationfactor XII (fXII) are protected from arterial thrombosis and cerebral ischemia-reperfusion injury. We demonstrate that selective reduction of prekallikrein (PKK), another member of the contact system, using antisense oligonucleotide (ASO) technology results in an antithrombotic phenotype in mice. The effects of PKK deficiency were compared with those of fXII deficiency produced by specific ASO-mediated reduction of fXII. Mice with reduced PKK had ∼ 3-fold higher plasma levels of fXII, and reduced levels of fXIIa-serpin complexes, consistent with fXII being a substrate for activated PKK in vivo. PKK or fXII deficiency reduced thrombus formation in both arterial and venous thrombosis models, without an apparent effect on hemostasis. The amount of reduction of PKK and fXII required to produce an antithrombotic effect differed between venous and arterial models, suggesting that these factors may regulate thrombus formation by distinct mechanisms. Our results support the concept that fXII and PKK play important and perhaps nonredundant roles in pathogenic thrombus propagation, and highlight a novel, specific and safe pharmaceutical approach to target these contact system proteases.

Recent studies indicate that the plasma contact system plays an important role in thrombosis, despite being dispensable for hemostasis. For example, mice deficient in coagulationfactor XII (fXII) are protected from arterial thrombosis and cerebral ischemia-reperfusion injury. We demonstrate that selective reduction of prekallikrein (PKK), another member of the contact system, using antisense oligonucleotide (ASO) technology results in an antithrombotic phenotype in mice. The effects of PKK deficiency were compared with those of fXII deficiency produced by specific ASO-mediated reduction of fXII. Mice with reduced PKK had ∼ 3-fold higher plasma levels of fXII, and reduced levels of fXIIa-serpin complexes, consistent with fXII being a substrate for activated PKK in vivo. PKK or fXII deficiency reduced thrombus formation in both arterial and venous thrombosis models, without an apparent effect on hemostasis. The amount of reduction of PKK and fXII required to produce an antithrombotic effect differed between venous and arterial models, suggesting that these factors may regulate thrombus formation by distinct mechanisms. Our results support the concept that fXII and PKK play important and perhaps nonredundant roles in pathogenic thrombus propagation, and highlight a novel, specific and safe pharmaceutical approach to target these contact system proteases. PMID:21821705

Nanofiltration assures that protein therapeutics are free of adventitious agents such as viruses. Nanofilter pores must allow passage of protein drugs but be small enough to retain viruses. Five nanofilters have been evaluated to identify those that can be used interchangeably to yield a high purity CoagulationFactor IX product. When product preparations prior to nanofiltration were analyzed using electrophoresis, Western blot, liquid chromatography - tandem mass spectrometry and size exclusion HPLC, factor IX, inter - α - trypsin inhibitor and C4b binding protein (C4BP) were observed. C4BP was removed from product by all five nanofilters when nanofiltration was performed at physiological ionic strength. However, at high ionic strength, C4BP was removed by only two nanofilters. HPLC indicated that the Stokes radius of C4BP was larger at low ionic strength than at high ionic strength. The results suggest that C4BP exists in an open conformation at physiological ionic strength and is removed by nanofiltration whereas, at high ionic strength, the protein collapses to an extent that allows passage through some nanofilters. Manufacturers should be aware that protein contaminants in other nanofiltered protein drugs could behave similarly and conditions of nanofiltration must be evaluated to ensure consistent product purity.

This review describes some natural proteins, which can be employed, either as factor concentrates derived from human plasma or as recombinant drug, to modulate the coagulation system. I will address some biochemical characteristics and the physiological role of von Willebrand factor, the coagulationfactors of the extrinsic and intrinsic pathways, and the physiological anticoagulant protein C. In addition, I will detail the pharmacological compounds, which are available for influencing or substituting the coagulation proteins: desmopressin (DDAVP), single coagulationfactor concentrates, prothrombin complex concentrates, and protein C concentrate. In particular, I will address some treatment topics of general medical interest, such as the treatment of massive bleeding, the correction of the coagulopathy induced by vitamin K-antagonists in patients with cerebral haemorrhage, and of the coagulopathy of meningococcemia. Finally, I will describe some properties and practical clinical applications of the recombinant anticoagulans lepirudin and bivalirudin, which are derived from hirudin, the natural anticoagulant of the medical leech.

Deinagkistrodon acutus venom contains a collection of anticoagulant proteins that has been reported to prevent prothrombinase assembly (Teng and Seegers, 1981, Thromb. Res. 23, 255). A partial sequence indicates that these proteins are related to the functionally equivalent protein in Trimeresurus flavoviridis (Atoda et al., 1991, J. Biochem. 106, 808). Inhibition of prothrombinase, the complex of Factors Xa and Va combined with phospholipids, is expressed in bovine, human, and rat plasmas as indicated by an assay dependent on only prothrombinase activity. The concentration dependence of inhibition of prothrombin conversion by different combinations of the components of bovine prothrombinase under the same conditions yielded estimates of apparent dissociation constants of 104 nM and 2 nM for complexes of the inhibitor with Factor Xa and with Factors Xa and Va, respectively. Because this inhibitor does not prevent Factor Xa alone from converting prothrombin, but blocks the other combinations, we conclude the inhibitor prevents the complex of Factors Xa and Va from binding to phospholipid surfaces and to prothrombin. The inhibitor also blocks the activation of Factor X by Factor VIIa and thromboplastin as well. However, the inhibitor has no effect on thrombin-induced clotting or fibrinolysis induced by either plasminogen activator or streptokinase. Therefore, this inhibitor has several properties required of an anticoagulant, therapeutic agent.

Sheehan's Syndrome (SS) is defined as pituitary hormone deficiency due to ischemic infarction of the pituitary gland as a result of massive postpartum uterine hemorrhage. Herein, we aimed to investigate the roles of Factor II (G20210A), Factor V (G1691A), MTHFR (C677T and A1298C), PAI-1 4G/5G, and TNF-α (−308 G > A) gene polymorphisms in the etiopathogenesis of SS. Venous blood samples were obtained from 53 cases with SS and 43 healthy women. Standard methods were used to extract the genomic DNAs. Factor II (G20210A), Factor V (G1691A), and MTHFR (C677T and A1298C) polymorphisms were identified by real-time PCR. PAI-1 4G/5G and TNF-α (−308 G > A) gene polymorphisms were detected with polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods. According to statistical analysis, none of the polymorphisms were found to be significantly higher in the SS group compared to the control group. Hence, we suggest that genetic factors other than Factor II, Factor V, MTHFR, PAI-1, and TNF-α gene polymorphisms should be researched in the etiopathogenesis of SS. PMID:24891849

Sheehan's Syndrome (SS) is defined as pituitary hormone deficiency due to ischemic infarction of the pituitary gland as a result of massive postpartum uterine hemorrhage. Herein, we aimed to investigate the roles of Factor II (G20210A), Factor V (G1691A), MTHFR (C677T and A1298C), PAI-1 4G/5G, and TNF- α (-308 G > A) gene polymorphisms in the etiopathogenesis of SS. Venous blood samples were obtained from 53 cases with SS and 43 healthy women. Standard methods were used to extract the genomic DNAs. Factor II (G20210A), Factor V (G1691A), and MTHFR (C677T and A1298C) polymorphisms were identified by real-time PCR. PAI-1 4G/5G and TNF- α (-308 G > A) gene polymorphisms were detected with polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods. According to statistical analysis, none of the polymorphisms were found to be significantly higher in the SS group compared to the control group. Hence, we suggest that genetic factors other than Factor II, Factor V, MTHFR, PAI-1, and TNF- α gene polymorphisms should be researched in the etiopathogenesis of SS.

Previous studies demonstrated that several normal and transformed cultured human cell lines specifically bind human coagulationfactors VII and VIIa via tissue factor. In the present study, we show that 125I-radiolabeled recombinant human factor VIIa (125I-rFVIIa) binds to a human hepatoma cell line (HuH7). In the presence of rabbit polyclonal anti-human tissue factor apoprotein IgG, binding of 125I-rFVIIa to the HuH7 cells was decreased approximately 60%, suggesting of tissue factor-independent binding sites for 125I-rFVIIa on these cells. The binding isotherm of 125I-rFVIIa for the HuH7 cells in the presence of anti-tissue factor IgG exhibited a hyperbolic profile and was time-, temperature- and calcium-dependent. Furthermore, binding at 4 degrees C was specific, dose-dependent and saturable. Scatchard analysis of the binding data demonstrated a single class of binding sites with a dissociation constant (Kd) of 3.2 nM and 27,000 binding sites per cell. At 4 degrees C, 125I-rFVIIa bound to, and eluted from, the cell was indistinguishable from offered 125I-rFVIIa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. The molecular properties of the tissue factor-independent binding protein were studied by using the cleavable cross-linking agent 3,3'-dithiobis(sulfosuccinimidylpropionate). A cross-linking product of 125I-rFVIIa and a cell surface protein with an apparent M(r) approximately 100,000 was observed. The cross-linking reaction was strongly inhibited by a 100-fold molar excess of unlabeled rFVIIa, but not by rabbit polyclonal anti-human tissue factor apoprotein IgG, indicating that cross-linking does not involve the extracellular domain of tissue factor.(ABSTRACT TRUNCATED AT 250 WORDS)

In 2010-2012, the North American Specialized Coagulation Laboratory Association (NASCOLA) distributed 12 proficiency testing challenges to evaluate laboratory testing for protein S (PS). Results were analysed to assess the performance of PS activity, PS free antigen, and PS total antigen testing. Statistical analysis was performed on the numeric results and qualitative classification submitted for each method. There were 2,106 total results: 716 results from PS activity assays, 833 results from PS free antigen assays, and 557 results from PS total antigen assays. The three assay types performed well in the classification of five normal samples and nine abnormal samples, although certain PS activity methods were more likely to classify normal samples as abnormal and one PS total antigen assay was more likely to classify abnormal samples as normal. PS activity methods were affected by interfering substances such as heterozygous or homozygous factor V Leiden mutation (underestimation) and the anticoagulant drug rivaroxaban (overestimation). In conclusion, NASCOLA laboratories using a variety of PS assays performed well in the classification of clearly normal and abnormal samples. Laboratories performing PS activity assays should be aware of potential interferences in samples positive for FV Leiden or containing certain anticoagulant medications.

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated human recombinant factor VIII for the treatment of hemophilia A. The safety profile of rFVIII, and polyethylene glycols (PEG) technology, is well-established. Conducting long-term toxicity studies in animals using human proteins can be complicated by anti-drug antibody (ADA) development. To evaluate long-term safety of N8-GP, 26- and 52-week toxicity studies were conducted in immune-deficient rats dosed intravenously every fourth day with 0, 50, 150, 500, or 1200 IU/kg N8-GP. Observations included clinical observations, body weight, ophthalmoscopy, hematology, chemistry, coagulation, urinalysis, toxicokinetics, antibody analysis, and macroscopic/microscopic organ examination. Immunohistochemical staining examined the distribution of PEG in the brain. No adverse test item-related findings were seen and PEG was not detected in the brain. Exposure was confirmed for ~75% of the animals dosed with 500 and 1200 IU/kg N8-GP; the high lower limit of quantification of the bioanalysis assay prevented confirmation of exposure in the lower doses. A small number of animals developed ADAs, and the proportion of animals surviving until scheduled termination was >80%. N8-GP was well tolerated, and the immune-deficient rat proved suitable for testing long-term toxicity of human proteins that are immunogenic in animals. PMID:28458688

Antibodies (inhibitors) developed by hemophilia B patients against coagulationfactor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ∼2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP(+) regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ∼870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft(2) per annum yielding 24,000-36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs.

The R506Q point mutation in the gene coding for coagulationfactor V (Leiden mutation) is the major underlying defect in resistance to activated protein C (APC), which predisposes to venous thrombosis. The risk of deep vein thrombosis is clearly elevated in carriers of the mutation, but the risk for pulmonary embolism has not been demonstrated to be as high. The aim of our study was to determine the frequency of the Leiden mutation in an autopsy series of sudden fatal pulmonary embolism cases. PCR and subsequent restriction enzyme digestion were applied for genotyping 164 cases of pulmonary embolism. According to our data, the allele frequency of the Leiden mutation is not higher in sudden fatal pulmonary embolism cases (0.8%, 95% CI 0-1.9%) than in the general Finnish population (1.5%, 95% CI 0-3.3%). In addition to the 97 Finns, we determined the frequency of the Leiden mutation in 255 individuals from the neighbouring populations (Saami, Komi, and Karelians from Russia and Estonians), and found the Saami to have the highest frequency of the Leiden mutation (6.3%, 95% CI 3.2-9.2) in the general northern European population sample studied here.

Arterial thrombosis is an important complication of diabetes and cancer, being an important target for therapeutic intervention. Crataeva tapia bark lectin (CrataBL) has been previously shown to have hypoglycemiant effect and also to induce cancer cell apoptosis. It also showed inhibitory activity against Factor Xa (Kiapp=8.6 μm). In the present study, we evaluated the anti-thrombotic properties of CrataBL in arterial thrombosis model. CrataBL prolongs the activated partial thromboplastin time on human and mouse plasma, and it impairs the heparin-induced potentiation of antithrombin III and heparin-induced platelet activation in the presence of low-dose ADP. It is likely that the dense track of positive charge on CrataBL surface competes with the heparin ability to bind to antithrombin III and to stimulate platelets. In the photochemically induced thrombosis model in mice, in the groups treated with 1.25, 5.0, or 10 mg/kg CrataBL, prior to the thrombus induction, the time of total artery occlusion was prolonged by 33.38%, 65%, and 66.11%, respectively, relative to the time of the control group. In contrast to heparin, the bleeding time in CrataBL-treated mice was no longer than in the control. In conclusion, CrataBL was effective in blocking coagulation and arterial thrombus formation, without increasing bleeding time.

Antibodies (inhibitors) developed by hemophilia B patients against coagulationfactor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (~1mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ~2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP+ regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ~870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft2 per annum yielding 24,000–36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs. PMID:26302233

Human coagulationfactor VII (FVII) has two N-glycosylation sites (N145 and N322) and two O-glycosylation sites (S52 and S60). In transiently transfected COS-7 cells, all combinations of N- and O-glycosylation knock-out mutations reduced the release of FVII to the medium. Pulse-chase analysis of CHO-K1 cell lines expressing recombinant FVII demonstrated that virtually all wild-type FVII synthesized was secreted from the cells, whereas both N- and O-glycosylation knock-out mutations induced partial intracellular degradation of the synthesized FVII. Likewise, two thirds of the FVII synthesized in vitamin K-depleted and warfarin-treated CHO cells was degraded intracellularly, demonstrating the importance of gamma-carboxylation for the secretion of FVII. The furin inhibitor decanoyl-R-V-K-R-chloromethylketone inhibited propeptide cleavage, but FVII with propeptide appeared to be secreted equally well as FVII without propeptide. Propeptide cleavage was not inhibited by vitamin K depletion and warfarin treatment, suggesting that for FVII, correct gamma-carboxylation is not required for optimal processing of the propeptide. In conclusion, all post-translational modifications of FVII except propeptide cleavage were important for complete secretion of the synthesized FVII and to avoid intracellular degradation. Thus, the extensive post-translational modification of FVII seems critical for the intracellular stability of the protein and is required for keeping the protein in the secretory pathway.

Hemostasis is traditionally defined as a physiological response to blood vessel injury and bleeding, which entails a co-ordinated process involving the blood vessel, platelets, and blood clotting proteins (i.e. coagulationfactors). Hemostasis can be divided into primary and secondary components. The former rapidly initiates after endothelial damage and is characterized by vascular contraction, platelet adhesion, and formation of a soft aggregate plug. The latter is initiated following the release of tissue factor and involves a complex sequence of events known as the blood coagulation cascade, encompassing serial steps where each coagulationfactor activates another in a chain reaction that culminates in the conversion of fibrinogen to fibrin. Patients carrying abnormalities of the coagulation cascade (i.e. deficiencies of coagulationfactors) have an increased bleeding tendency, where the clinical severity is mostly dependent upon the type and the plasma level of the factor affected. These disorders also impose a heavy medical and economic burden on individual patients and society in general. The aim of this article is to provide a general overview on the pathophysiology, clinics, diagnostics, and therapy of inherited disorders of coagulationfactors.

Isolated traumatic subarachnoid hemorrhage (itSAH) is found in approximately 25% of all patients with mild traumatic brain injury (TBI). The aim of this study was to analyze the clinical course and identify risk factors for potential clinical and radiologic deterioration in consideration of impaired coagulation in patients with itSAH. A retrospective analysis of 735 patients with TBI resulting in a pathologic computer-assisted tomography (CAT) was performed. Only those patients with itSAH and Glasgow Coma Scale (GCS) of greater than 8 points and follow-up CAT scan were included. Patients with hemorrhage in any other brain compartment (subdural, epidural, and intracerebral) were excluded. Impaired coagulation was operationally defined. Of the 735 patients, 89 met the inclusion criteria. The majority of these patients experienced mild TBI. The rate of radiologic expansion or conversion of the SAH was 28.1%. The rate of clinical deterioration was 6.7%. Neither the initial pattern of itSAH on different intracranial localizations nor the number of sulci involved in the itSAH was associated with clinical worsening. The rate of patients with impaired coagulation was 38%; 17.9% of all patients showed elevated international normalized ratio (INR). Radiologic and clinical deterioration was significantly associated with elevated INR. INR was shown to be independent of age in a logistic regression analysis. TBI patients with itSAH and impaired coagulation especially those who showed elevated INR are at risk of clinical and radiologic deterioration. Despite coagulation status, routine repetition of cranial CAT scan is advised in patients with itSAH to detect potential radiologic worsening, which if occurring should result in close clinical monitoring. Therapeutic study, level IV; prognostic study, level III.

The effect of 2 different practices for preparation of working dilutions in the chromogenic substrate method for potency assay of factor VIII was evaluated. In this study the potency of several concentrate materials was shown to be statistically equivalent, whether performing the assay with independent or serial working dilutions.

Background EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7, a causative agent of diarrhea-associated Hemolytic Uremic Syndrome (D+HUS). The mechanism by which EHEC induces D+HUS has not been fully elucidated. Objectives We investigated the effects of EspP on clot formation and lysis in human blood. Methods Human whole blood and plasma were incubated with EspPWT at various concentrations and sampled at various time points. Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT), coagulationfactor activities, and thrombelastgraphy (TEG) were measured. Results and Conclusions Human whole blood or plasma incubated with EspPWT was found to have prolonged PT, aPTT, and TT. Furthermore, human whole blood or plasma incubated with EspPWT had reduced activities of coagulationfactors V, VII, VIII, and XII, as well as prothrombin. EspP did not alter the activities of coagulationfactors IX, X, or XI. When analyzed by whole blood TEG, EspP decreased the maximum amplitude of the clot, and increased the clot lysis. Our results indicate that EspP alters hemostasis in vitro by decreasing the activities of coagulationfactors V, VII, VIII, and XII, and of prothrombin, by reducing the clot strength and accelerating fibrinolysis, and provide further evidence of a functional role for this protease in the virulence of EHEC and the development of D+HUS. PMID:26934472

This report summarizes recommendations relating to haemophilia therapy arising from discussions among experts from 36 European countries during the Kreuth III meeting in April 2013. To optimize the organization of haemophilia care nationally, it is recommended that a formal body be established in each country to include the relevant clinicians, national haemophilia patient organization, health ministry, paying authority and (if appropriate) regulatory authorities. The minimum factor VIII consumption level in a country should be 3 I.U. per capita. Decisions on whether to adopt a new product should not be based solely on cost. Prophylaxis for children with severe haemophilia is already recognized as the optimum therapy. Ongoing prophylaxis for individual adults should also be provided when required based on clinical decision making by the clinician in consultation with the patient. Children with inhibitors who have failed, or who are not suitable for, immune tolerance therapy should be offered prophylaxis with bypassing agents. Single factor concentrates should be used as therapy wherever possible in patients with rare bleeding disorders. Orphan drug designation for a factor concentrate should not be used to hinder the development, licencing and marketing of other products for the same condition which have demonstrably different protein modification or enhancement.

Summary A global phase 3 study evaluated the pharmacokinetics, efficacy and safety of a recombinant fusion protein linking coagulationfactor IX with albumin (rIX-FP) in 27 previously treated male children (1–11 years) with severe and moderately severe haemophilia B (factor IX [FIX] activity ≤2 IU/dl). All patients received routine prophylaxis once every seven days for up to 77 weeks, and treated any bleeding episodes on-demand. The mean terminal half-life of rIX-FP was 91.4 hours (h), 4.3-fold longer than previous FIX treatment and clearance was 1.11 ml/h/kg, 6.4-fold slower than previous FIX treatment. The median (Q1, Q3) annualised spontaneous bleeding rate was 0.00 (0.00, 0.91) and was similar between the <6 years and ≥6 years age groups, with a weekly median prophylactic dose of 46 IU/kg. In addition, patients maintained a median trough level of 13.4 IU/dl FIX activity on weekly prophylaxis. Overall, 97.2% of bleeding episodes were successfully treated with one or two injections of rIX-FP (95% CI: 92% to 99%), 88.7% with one injection, and 96% of the treatments were rated effective (excellent or good) by the Investigator. No patient developed FIX inhibitors and no safety concerns were identified. These results indicate that rIX-FP is safe and effective for preventing and treating bleeding episodes in children with haemophilia B with weekly prophylaxis. Routine prophylaxis with rIX-FP at treatment intervals of up to 14 days are currently being investigated in children with severe and moderately severe haemophilia B. Clinicaltrials.gov (NCT01662531) PMID:27583313

Congenital factor VII (FVII) and factor X (FX) deficiencies belong to the group of rare bleeding disorders which may occur in separate or combined forms since both the F7 and F10 genes are located in close proximity on the distal long arm of chromosome 13 (13q34). We here present data of 192 consecutive index cases with FVII and/or FX deficiency. 10 novel and 53 recurrent sequence alterations were identified in the F7 gene and 5 novel as well as 11 recurrent in the F10 gene including one homozygous 4.35 kb deletion within F7 (c.64+430_131-6delinsTCGTAA) and three large heterozygous deletions involving both the F7 and F10 genes. One of the latter proved to be cytogenetically visible as a chromosome 13q34 deletion and associated with agenesis of the corpus callosum and psychomotor retardation. Large deletions play a minor but essential role in the mutational spectrum of the F7 and F10 genes. Copy number analyses (e. g. MLPA) should be considered if sequencing cannot clarify the underlying reason of an observed coagulopathy. Of note, in cases of combined FVII/FX deficiency, a deletion of the two contiguous genes might be part of a larger chromosomal rearrangement.

Summary Background Heterologous expression of Factor VIII (FVIII) is about 2 to 3 orders of magnitude lower than similarly sized proteins. Bioengineering strategies aimed at different structural and biochemical attributes of FVIII have been successful in enhancing its expression levels. Objective Disulfide bonds are vital to the proper folding, secretion and stability of most secretory proteins. In an effort to explore additional targeted bioengineering approaches, the role of disulfide bonds in FVIII secretion and function was probed in this study. Methods and Results Single and paired cysteine mutants were generated by substituting with serine or glycine residues and analyzed by transient transfection into COS-1 and CHO cells. Seven of the eight disulfide bonds in FVIII were found to be indispensable for proper secretion and function. However, elimination of the disulfide bond formed by C1899 and C1903 within the conserved A3 domain improved the secretion of FVIII. The addition of the C1899G/C1903G mutations to a previously described FVIII variant, 226/N6, with high secretion efficiency increased its secretion by 2.2-fold. Finally, the addition of the A1-domain mutation, F309S in conjunction with the disulfide mutation had an additive effect resulting in a net improvement in secretion of between 35–45 fold higher than wild type FVIII in CHO cells. Conclusion Such combined targeted bioengineering strategies may facilitate more efficient production of recombinant FVIII toward low cost factor replacement therapy for hemophilia A. PMID:22044596

Accurate computer simulation of blood function can inform drug target selection, patient-specific dosing, clinical trial design, biomedical device design, as well as the scoring of patient-specific disease risk and severity. These large-scale simulations rely on hundreds of independently measured physical parameters and kinetic rate constants. However, the models can be validated against large scale, patient-specific laboratory measurements. By validation with high dimensional data, modelling becomes a powerful tool to predict clinically complex scenarios. Currently, it is possible to accurately predict the clotting rate of plasma or blood in a tube as it is activated with a dose of tissue factor, even as numerous coagulationfactors are altered by exogenous attenuation or potentiation. Similarly, the dynamics of platelet activation, as indicated by calcium mobilisation or inside-out signalling, can now be numerically simulated with accuracy in cases where platelets are exposed to combinations of agonists. Multiscale models have emerged to combine platelet function and coagulation kinetics into complete physics-based descriptions of thrombosis under flow. Blood flow controls platelet fluxes, delivery and removal of coagulationfactors, adhesive bonding, and von Willebrand factor conformation. The field of Blood Systems Biology has now reached a stage that anticipates the inclusion of contact, complement, and fibrinolytic pathways along with models of neutrophil and endothelial activation. Along with “-omics” data sets, such advanced models seek to predict the multifactorial range of healthy responses and diverse bleeding and clotting scenarios, ultimately to understand and improve patient outcomes. PMID:23809126

Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.

The naturally occurring M358R mutation of the plasma serpin α1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg-Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg-Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10(2) M(-1)sec(-1). We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin.

In the previous studies, some authors reported that automated apheresis leads to a hypercoagulable state. We tried to find out changes in coagulation parameters after double plateletpheresis in this study. Forty-five donors were recruited to the study, and coagulation parameters were assessed before and after double plateletpheresis. After double plateletpheresis, fibrinogen, factor V, factor VIII and factor IX were decreased compared with the values before apheresis. Although serum levels of this coagulation parameters are decreasing, they are still in the normal limits. Therefore, we suggest that double plateletpheresis is a safe procedure for healthy volunteers taking into account these coagulation parameters.

The performance of factor VII (FVII) assays currently used by clinical laboratories was examined in North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency tests. Data from 12 surveys conducted between 2008 and 2010, involving 20 unique specimens plus four repeat-tested specimens, were analyzed. The number of laboratories per survey was 49-54 with a total of 1224 responses. Numerous reagent/instrument combinations were used. For FVII > 80 or <40 U/dL, 99.5% of results (859/863) were correctly classified by laboratories as normal/abnormal. Classification of specimens with 40-73 U/dL FVII was heterogeneous. Interlaboratory precision was better for normal specimens (coefficient of variation (CV) 10.7%) than for FVII<20 U/dL (CV 33.1%), with a mean CV of 17.2% per specimen. Intralaboratory precision for repeated specimens demonstrated no significant difference between the paired survey results (mean absolute difference 2.5-5.0 U/dL). For specimens with FVII >50 U/dL, among commonly used methods, one thromboplastin and one calibrator produced results 5-6 U/dL higher and another thromboplastin and calibrator produced results 5-6 U/dL lower than all other methods, and human thromboplastin differed from rabbit by +7.6 U/dL. Preliminary evidence suggests these differences could be due to the calibrator. For FVII <50 U/dL, differences among the commonly used reagents and calibrators were generally not significant.

To assess the hemostatic efficacy of recombinant coagulationfactor VIIa (rFVIIa) in the management of uncontrolled bleeding in postcardiac surgery with cardiopulmonary bypass in children. An open-label study. A postoperative intensive care unit. Eight consecutive pediatric patients with excessive bleeding after cardiac surgery with cardiopulmonary bypass that met the criteria for reexploration and did not respond to optimal transfusions of platelets and fresh frozen plasma. rFVIIa 30 microg/kg was given as a bolus injection. A higher dose of 60 microg/kg was used if a patient had preoperative coagulopathy, preoperative multiple-organ failure, or indications that required an emergency operation. The same dose was repeated 15 mins after the previous injection if the bleeding had not decreased. If the bleeding had decreased but still exceeded 10 mL/hr for body weight =5 kg or exceeded 2 mL.kg(-1).hr(-1) for body weight >5 kg, the same dose was repeated 2 hrs after the previous injection. A maximum of four doses could be given before rFVIIa was considered ineffective and a reexploration was needed. Postoperative blood loss was estimated from the volume of chest tube drainage. rFVIIa successfully controlled bleeding and prevented reexploration in all seven patients who received treatment according to the protocol. One patient who received only one dose of rFVIIa required reexploration because a second dose was not available. No adverse events related to rFVIIa were seen. rFVIIa may be useful in preventing reexploration in uncontrolled postoperative bleeding in children undergoing cardiac surgery with cardiopulmonary bypass. Randomized, placebo-controlled studies are needed to confirm the safety and efficacy of rFVIIa in this clinical setting.

Plasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measured-genotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL−1 (P = .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3′ splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant. PMID:17209060

To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulationfactor IX fused with cholera toxin β-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20–25% of control animals survived after six to eight F.IX doses, 90–93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyer's patches in the ileum. Within 2–5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5–80 μg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (∼40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment. PMID:20351275

Plasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measured-genotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL(-1) (P = .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3' splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant.

Previously we have characterized a binding site for high M(r) kininogen in the first of four tandem-repeat (Apple) domains within the heavy chain region of factor XI (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154; Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 267, 4247-4252), whereas a substrate binding site for factor IX was localized to the second Apple (A2) domain (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 266, 24190-24197). To define the factor XI domain that binds factor XIIa, we have screened a panel of synthetic peptides for their capacity to inhibit factor XI activation by factor XIIa. Peptide Gly326-Lys357 (located in the A4 domain) is a noncompetitive inhibitor of factor XI activation by factor XIIa (Ki = 3.75 microM), whereas structurally similar peptides from the A1, A2, and A3 domains were required at > 1000-fold higher concentrations for similar effects. The same peptide (Gly326-Lys357) is a competitive inhibitor of factor XIIa amidolytic activity (Ki = 3.8 microM) suggesting that it binds near the active site of factor XIIa. Computer modeling was used to predict the secondary and tertiary structure of the A4 domain of factor XI that interacts with factor XIIa. Rationally designed, conformationally constrained peptides were synthesized comprising residues Ala317-Gly326, Lys331-Lys340, and Gly344-Gly350, which act in concert to inhibit factor XI-activation by factor XIIa. Finally, a conformationally constrained peptide spanning residues Ala317-Gly350 inhibits factor XIIa-catalyzed factor XI activation 50% at a concentration of 5 x 10(-7) M. These results, interpreted in the context of the model, suggest that the sequence of amino acids from Ala317 through Gly350 of the heavy chain of the A4 domain of factor XI contains three peptide structures, possibly consisting of three antiparallel beta-strands that together comprise a contact surface for interacting with

To further elucidate the connection between inflammation and factor VII (FVII) taking genetic variation in the FVII locus into account, we have examined 387 patients after myocardial infarction and 387 age-matched and sex-matched healthy control individuals. Circulating levels of C-reactive protein, FVII antigen (FVIIag), activated FVII (FVIIa), fibrinogen and interleukin-6 were analysed and all subjects were genotyped for the Arg353Gln polymorphism in the FVII locus. Plasma concentrations of C-reactive protein, fibrinogen, and interleukin-6 were higher among patients than control individuals. FVIIag was lower in the patient group, but for FVIIa there was no difference between the two groups. Among the inflammatory markers, only C-reactive protein indicated a weak nonlinear association with FVII. No significant difference in frequency of the Gln allele was observed between patients and control individuals but the presence of the Gln allele was associated with lower plasma levels of FVIIag and FVIIa in both groups. The low-grade chronic inflammation seen 3 months after myocardial infarction is not of major importance for the variation in plasma concentration of FVII. The presence of the Gln allele in the Arg353Gln polymorphism in the FVII locus did not differ between patients and control individuals but was associated with lower plasma levels of FVIIag and FVIIa that could have a protective effect against myocardial infarction. To further elucidate these facts, a prospective study should be performed to reduce the risk of a possible selection bias due to coronary heart disease death seen in retrospective case-control studies.

Background The snake venom group IIA secreted phospholipases A2 (SVPLA2), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA2-inhibitors from snake serum; and (iii) coagulationfactors present in human blood. Results Using surface plasmon resonance (SPR) protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulationfactor Xa (FXa) via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS). The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors. PMID:18062812

Inflammation and coagulation constitute two host defense systems with complementary roles in eliminating invading pathogens, limiting tissue damage, and restoring homeostasis. Extensive cross talk exists between these 2 systems, whereby inflammation leads to activation of coagulation, and coagulation considerably affects inflammatory activity. Infection leads to the production of proinflammatory cytokines that, in turn, stimulate the production of tissue factor. Activation of the coagulation system and ensuing thrombin generation are dependent on the expression of tissue factor. Conversely, activated coagulation proteases may affect specific receptors on inflammatory cells and endothelial cells and thereby modulate the inflammatory response. Activation of coagulation with the simultaneous down-regulation of endothelial-bound anticoagulant mechanisms and endogenous fibrinolysis characterizes the pathophysiology of sepsis. The mechanisms by which these highly complex and codependent defense strategies are linked together both in health and disease is the focus of this review.

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Background: Fresh frozen plasma (FFP) is considered adequate for transfusion immediately after thawing or for up to 24 hours if kept at 1–6°C, and is currently used very often to replace deficient clotting factors. If factor levels in refrozen FFP are within normal limits, then this component can possibly be transfused, thus avoiding wastage of FFP. Aim: To study the fate of vitamin K-dependent coagulationfactors (F II, F VII, F IX, F X) and fibrinogen activity levels in repeatedly (twice) frozen and thawed FFP. Materials and Methods: Two hundred FFP units comprising 50 units of each major blood group (A, B, AB, and O) were thawed at 37°C and 10–20 mL of FFP transferred to transfer bags with the help of a sterile connecting device (SCD). The FFP samples were taken into tubes (first sampling), and then the transfer bags were kept for 24 hours at 4°C. After 24 hours, repeat samples were taken in tubes from the transfer bag (second sampling), and then the bags were re-stored at < -18°C. One week later, the above procedure was repeated. Activity of coagulationfactors and fibrinogen levels were measured by the automated coagulation analyzer. Results: The levels of F II, F VII, F IX, F X, and fibrinogen of all the 200 FFP units, at all four time points, were above the lower normal value, but well within the normal range. Conclusion: The levels of F II, F VII, F IX, F X, and fibrinogen remain stable and adequate for transfusion in twice-thawed-and-refrozen FFP. This component can be safely used for transfusion as a source of vitamin K-dependent clotting factors and fibrinogen. PMID:23559757

Modified ultrafiltration (MUF) is a technique that hemoconcentrates residual CPB circuit blood and the patient at the same time. Hemoconcentration and MUF are Class 1-A recommendations in the anesthesia and surgical blood conservation guidelines. This study evaluated the off-line MUF process of the Hemobag (HB, Global Blood Resources, Somers, CT, USA) to quantitate coagulationfactor levels, platelet (PLT) count and function in one facility and cellular growth factor concentrations of the final product that were transfused to the patient in another facility In two cardiac surgery facilities, after decannulation, the extracorporeal circuit (ECC) blood from 22 patients undergoing cardiac surgery was processed with the HB device. In eleven patients from the first facility by the study design, blood samples for coagulationfactor levels and PLT aggregation were drawn from the reservoir of the MUF device pre- and post-processing. The samples (n = 11) were sent to a reference laboratory where testing for prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), reptilase time, fibrinogen, clotting factors II, V, VII, VIII, IX, X, ADAMTS-13, protein C, protein S, antithrombin III, von Willebrand Factor (vWF), and platelet (PLT) aggregation were performed. A portion of the final concentrated HB blood samples (n = 5-10) from the second facility by design were evaluated for transforming and platelet-derived cellular growth factor concentrations. On average, approximately 800 - 2000 mls of whole blood were removed from the ECC post-CPB for processing in the HB device. After processing, there was, on the average, approximately 300 - 950 mls of concentrated whole blood salvaged for reinfusion. The PT and INR were significantly lower in the post-processing product compared to the pre-processing samples while the aPTT times were not significantly different. All coagulationfactors and natural anti-coagulants were significantly

Bypassing inhibitors in haemophilia patients is limited to activated (a) Factor(F)VII products. We introduced "FVa activity augmentation" as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilising properties of superFVa and recombinant human (rh)FVIIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in haemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. SuperFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e. g. 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalise clot lysis times could be reduced by 100-fold (e. g. from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In haemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of haemophilia with inhibitors and it merits further evaluation for new bypassing strategies.

Bypassing inhibitors in hemophilia patients is limited to activated (a) Factor(F)VII products. We introduced “FVa activity augmentation” as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilizing properties of superFVa and recombinant human (rh)FVIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in hemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. superFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e.g., 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalize clot lysis times could be reduced by 100-fold (e.g., from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In hemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of hemophilia with inhibitors and it merits further evaluation for new bypassing strategies. PMID:26466980

Studies of plasmas from individuals with Hageman trait (factor XII deficiency), plasma thromboplastin antecedent (PTA, factor XI) deficiency, Fletcher trait (plasma prekallikrein deficiency) and Fitzgerald trait (high molecular weight-kininogen deficiency) have revealed the importance of these proteins in blood coagulation. The interactions among them, however, are not fully elucidated. We have studied these reactions by two different approaches. (1) In a purified system, high molecular weight kininogen was absolutely required for activation of PTA by HF and ellagic acid (EA). The yield of activated PTA was proportional to the amount of HF, HMW-K, and PTA in the mixtures, suggesting that these three proteins may form a complex in the presence of EA. (2) In experiments with whole plasma, we took advantage of the adsorption of EA to Sephadex gels. When normal plasma or plasma deficient in HF, PK, HMW-K or PTA was exposed to Sephadex-EA and was separated by centrifugation, each supernatant plasma except that deficient in HF shortened the prolonged partial thromboplastin time (PTT) of HF-deficient plasma. Plasma simultaneously depleted of HMW-K, PK and PTA also shortened the PTT of HF-deficient plasma and of plasma depleted of HF and PK, but had virtually no procoagulant effect upon the PTT of plasma depleted of HF and MHW-K. Thus, exposure of HF in plasma to Sephadex-EA appeared to generate a clot-promoting form of HF in the absence of other clotting factors, but its expression required the presence of HMW-K.

von Willebrand factor (vWF) is a multimeric glycoprotein which functions within the coagulation system. It colocalizes with factor VIII (FVIII) by non-covalent interaction and alters its intracellular trafficking. vWF is also instrumental in maintaining the stability of secreted FVIII. The principal objective of this study was to generate a lentivirus-based vWF expression vector for gene therapy of hemophilia A. We inserted a vWF of 8.8 Kb into a lentiviral vector thereby producing VSV-G-pseudotyped vEx52. However, its titer was quite low, presumably because the length of vWF gene exceeds the size limit of the lentiviral vector. In order to overcome the low-titer, we concentrated the vEx52 and thus increased the efficiency of transduction approximately 6-fold with 1/100th of the volume. However, as concentration requires an additional laborious step, we attempted to enhance the transduction efficiency by deleting exons 24-46 and 29-46 in pRex52 to construct pRex23 and pRex28, and in pvEx52, yielding pvEx23 and pvEx28, respectively. The transfected pRex52 had a profound effect on the activity of secreted FVIII, and this activity declined as domains of vWF were deleted. However, when the domain-deleted vWF-lentiviruses were transduced into K562 cells, the vEx28 increased the activity of the secreted FVIII compared to what was observed with vEx52. This result is probably due to higher efficiencies of transduction and expression while retaining the essential domains required for proper interaction with FVIII.

Background Hereditary angioedema with normal C1-inhibitor and mutations in the coagulationfactor 12 gene is a recently described disease entity that occurs predominantly in women. Up to date, 2 different missense mutations of codon p.Thr328* in the coagulationfactor 12 gene have been reported, co-segregating with clinical signs. Aim of the study was to assess the clinical symptoms, mutations in the factor 12 gene, and plasma parameters of this disease in a family with hereditary angioedema with normal C1-inhibitor. Methods Six members of one family were studied, including 2 women with recurrent angioedema. Mutation analysis of the factor XII gene was performed. Results By sequencing the factor 12 gene, a hitherto unknown mutation, the deletion of 72 base pairs (c.971_1018+24del72*), was identified in a family of Turkish origin, in 2 women with recurrent skin swellings and abdominal pain attacks, and in their symptom-free father. The novel mutation c.971_1018+24del72* caused a loss of 48 base pairs of exon 9 (coding amino acids 324* to 340*) in addition to 24 base pairs of intron 9, including the authentic donor splice site of exon 9. All carriers of this mutation had normal plasma concentrations and activity of C1-inhibitor, C4, factor XII clotting activity, and activated partial thromboplastin times. Conclusions The novel deletion mutation in the factor 12 gene was located in the same F12 gene region as the missense mutations p.Thr328Lys* and p.Thr328Arg* reported previously, suggesting the importance of the exon 9 to intron 9 DNA region for the development of hereditary angioedema with normal C1-inhibitor.

In the majority of patients with severe sepsis, systemic activation of coagulation is present. Increasing evidence points to an extensive cross-talk between coagulation and inflammation that may play an important role in the pathogenesis of sepsis. Inflammation not only leads to activation of coagulation, but coagulation also considerably affects inflammatory activity. Molecular pathways that contribute to inflammation-induced activation of coagulation have been precisely identified. Proinflammatory cytokines and other mediators are capable of activating the coagulation system and downregulating important physiological anticoagulant pathways. Activation of the coagulation system and ensuing thrombin generation is dependent on expression of tissue factor on activated mononuclear cells and endothelial cells, and is insufficiently counteracted by TFPI. Simultaneously, endothelial-bound anticoagulant mechanism, in particular the protein C system, is shutoff by proinflammatory cytokines. In addition, fibrin removal is severely inhibited, because of inactivation of the fibrinolytic system, caused by an upregulation of its main inhibitor, plasminogen activator inhibitor type 1 (PAI-1). Increased fibrin formation and impaired removal lead to (micro)vascular thrombosis, which may result in tissue ischemia and subsequent organ damage. The cornerstone of the management of coagulation in sepsis is the specific and vigorous treatment of the underlying disorder. Strategies aimed at the inhibition of coagulation activation may theoretically be justified and have been found beneficial in experimental and initial clinical studies. Heparin may be an effective anticoagulant approach and alternative strategies comprise restoration of physiological anticoagulant pathways.

The prevention of blood coagulation is important in treating thromboembolic disorders, and several serine proteases involved in the coagulation cascade have been classified as pharmaceutically relevant. Whereas structure-based drug design has contributed to the development of some serine protease inhibitors, traditional computational methods have not been able to fully describe structure-activity relationships (SAR). Here, we study the SAR for a number of serine proteases by using a method that calculates the thermodynamic properties (enthalpy and entropy) of the water that solvates the active site. We show that the displacement of water from specific subpockets (such as S1-4 and the ester binding pocket) of the active site by the ligand can govern potency, especially for cases in which small chemical changes (i.e., a methyl group or halogen) result in a substantial increase in potency. Furthermore, we describe how relative binding free energies can be estimated by combining the water displacement energy with complementary terms from an implicit solvent molecular mechanics description binding.

Extensive surveys for the effects of various beta-D-glucans on the coagulation cascade in horseshoe crab amebocyte lysates showed that low-mol-wt-(1-->3)-beta-D-glucans and laminaran oligosaccharides inhibit the activation of a limulus coagulationfactor G by high-mol-wt-(1-->3)-beta-D-glucans. The inhibitory properties are exclusively dependent upon their number-average mol wt (Mn) in a range of 342-58,100, which correspond to a degree of polymerization (dp) range of 2-359. The most effective is a laminaran dextrin of Mn 5800 (dp of 35-36), which causes 50% inhibition of factor G activation at a concentration of 3.16 ng/mL. The inhibition of the activation of factor G proportional to the concentration of the inhibitor, and the adsorption of factor G by inhibitory beta-D-glucan-conjugated cellulose suggested a high affinity of the inhibitory saccharides for the activator-recognition site of factor G. Branched (1-->6), (1-->3)-beta-D-glucans, laminarans, mixed linkage (1-->3), (1-->4)-beta-D-glucans, and partially substituted curdlan and laminaran were found to be inhibitory, possibly owing to clusters of consecutive (1-->3)-beta-D-glucopyranosyl residues as intrachain units. The inhibition appears to be related to the inability of the inhibitory (1-->3)-beta-D-glucans to form ordered conformations and to their tendency to take a random-coil structure in aqueous solution.

Background ABO blood group is a hereditary factor of plasma levels of coagulationfactor VIII (FVIII) and von Willebrand factor (VWF). Age and gender have been shown to influence FVIII, VWF, fibrinogen (Fbg), and ADAMTS13 (A disintegrin and metalloprotease with thrombospondin type 1 motif, 13). We investigated the effects of ABO type, age, and gender on plasma levels of FVIII, Fbg, VWF, and ADAMTS13 in a Chinese population. Methods A total of 290 healthy volunteers were eligible for this study. ABO blood group was determined by indirect technique. FVIII:C and Fbg were measured by clotting assays. VWF antigen (VWF:Ag), collagen-binding activity (VWF:CBA), and ADAMTS13 antigen were assessed by ELISA, whereas VWF ristocetin cofactor activity (VWF:Rcof) was performed by agglutination of platelets with ristocetin. Results Mean FVIII:C and VWF levels (VWF:Ag, VWF:CBA, and VWF:Rcof) were significantly higher in non-O than in O type subjects (p

Critically ill patients are at a constant risk of direct (e.g., by pneumonia) or indirect lung injury (e.g., by sepsis). Excessive alveolar fibrin deposition is a prominent feature of lung injury, undermining pulmonary integrity and function. We examined the effect of local administration of recombinant human tissue factor pathway inhibitor (rh-TFPI), a natural anticoagulant, in two well-established models of lung injury in rats. Rats received intratracheal instillation of Pseudomonas aeruginosa, causing direct lung injury, or they received an intravenous injection of Escherichia coli lipopolysaccharide (LPS), causing indirect lung injury. Rats were randomized to local treatment with rh-TFPI or placebo through repeated nebulization. Challenge with P. aeruginosa or LPS was associated with increased coagulation and decreased fibrinolysis in bronchoalveolar lavage fluid (BALF) and plasma. Rh-TFPI levels in BALF increased after nebulization, whereas plasma rh-TFPI levels remained low and systemic TFPI activity was not affected. Nebulization of rh-TFPI attenuated pulmonary and systemic coagulation in both models, without affecting fibrinolysis. Nebulization of rh-TFPI modestly reduced the inflammatory response and bacterial growth of P. aeruginosa in the alveolar compartment. Local treatment with rh-TFPI does not alter systemic TFPI activity; however, it attenuates both pulmonary and systemic coagulopathy. Furthermore, nebulized rh-TFPI modestly reduces the pulmonary inflammatory response and allows increased bacterial clearance in rats with direct lung injury caused by P. aeruginosa.

The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.

We investigated the possibility that despite postoperative derangements of routine laboratory coagulation tests, markers of coagulation activation and thrombin generation would be normal or increased in patients undergoing hepatic resection for cancer In addition to the conventional coagulation tests prothrombin time and activated partial thromboplastin time, we measured select markers of coagulation activation prothrombin fragments 1 and 2 (PF1 + 2), thrombin-antithrombin complexes and plasma von Willebrand Factor antigen in 21 patients undergoing hepatic resection. The impact of hepatic resection on coagulation and fibrinolysis was studied with thromboelastography. Preoperatively, routine laboratory coagulation and liver function tests were normal in all patients. On the first postoperative day, prothrombin time was prolonged (range 16 to 22 seconds) in eight patients (38%). For these patients, thromboelastography was normal in six (75%), PF1 + 2 was elevated in four (50%), and thrombin-antithrombin complexes and von Willebrand Factor antigen were elevated in all, which was evidence of acute phase reaction, sustained coagulationfactor turnover and activation. By the fifth postoperative day, despite normalisation of prothrombin time, markers of increased coagulation activity remained greater than 85% of baseline values. The findings indicate that in patients undergoing liver resection for cancer, there is significant and prolonged postoperative activation of the haemostatic system despite routine coagulation tests being normal or even prolonged. Before considering therapeutic interventions an integrated approach to interpreting haematological data with clinical correlation is essential.

Immunoassays play a pivotal role in the clinical laboratory. In the coagulation section of the laboratory, they are used as an aid for diagnosis of deep vein thrombosis or pulmonary embolism, thrombophilia screening, or detection of coagulationfactor deficiencies, respectively. Enzyme-linked immunosorbent assay (ELISA) and latex agglutination immunoassay technologies are currently most widely used, while Luminescent Oxygen Channeling Immunoassay (LOCI®) and other chemiluminescence-based immunoassays are emerging technologies for the coagulation laboratory. However, not all immunoassay technologies employed are compatible with the workflow requirements of the coagulation laboratory, and, not all technologies are suitable for detection or quantification of every marker. This review focuses on technical and performance aspects of those immunoassay technologies that are most widely used in the coagulation laboratory, and provides a description of markers that are typically tested by immunoassays.

A new coagulant obtained through polymerization of Acacia mearnsii de Wild tannin extract has been characterized in the removal of two dangerous dye pollutants: Alizarin Violet 3R and Palatine Fast Black WAN. This coagulant is lab-synthesized according to the etherification of tannins with glycidyltrimethylammonium chloride and formaldehyde and its performance in dye removal in terms of efficiency was high. Reasonably low coagulant dosages (ca. 50 mg L-1) reaches high capacity levels (around 0.8 for Alizarin Violet 3R and 1.6 for Palatine Fast Black WAN mg dye mg-1 of coagulant) and pH and temperature are not extremely affecting variables. The systems coagulant dyes were successfully modeled by applying the Langmuir hypothesis. q max and b parameters were obtained with an adjusted correlation factor ( r 2) above 0.8.

Introduction Hemophilia A treatment involves replacing the deficient coagulationfactor VIII. This process may involve multiple steps that might create a barrier to adherence. A new dual-chamber syringe (DCS; FuseNGo®) was recently introduced with the aim of simplifying reconstitution. Aim This study aimed to identify factors associated with adult patients’ preferences for different coagulationfactor VIII reconstitution systems and to test ease of use and patient preference for the DCS. Methods A cross-sectional survey of adults with hemophilia A in five European countries was conducted; a subset of subjects also participated in a practical testing session of the DCS. Results Among the 299 survey participants, the device scenario requiring the least equipment and reconstitution steps (the DCS) received a median preference rating of 71 out of 100 (0 being “the least desirable” and 100 “the most desirable” rating). This was significantly higher than the other scenarios (the next highest achieved a median of 50 points; P<0.001). Participants would be more likely to use this device prophylactically (P<0.001). Among the 98 participants who tested the DCS, 57% preferred this device over their current device, 26% preferred their current device, and 17% had no preference. The DCS was rated as easier to use than current treatment devices (median score 9/10 versus 7/10 for current treatment, P=0.001). Conclusion The survey indicates that the prefilled DCS, FuseNGo®, requiring the least equipment and fewest reconstitution steps, was preferred by patients and was the device most likely to be used prophylactically; the practical device testing supports these results. PMID:25525348

Binding of coagulationfactors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulationfactors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow.

The effect of Ca2+, Mg2+, and Mn2+ on the initial rate of activation of human Factor X by the venom coagulant protein of Vipera russelli has been investigated. Neither Mg2+ nor Mn2+ alone support the reaction. Ca2+ is an essential activator and exhibits cooperative kinetics. Both Mg2+ and Mn2+ enhance the reaction cooperatively when Ca2+ is present at suboptimal concentrations. Similarly, Ca2+ quenches the intrinsic fluorescence of human Factor X in a cooperative manner. While neither Mg2+ nor Mn2+ by themselves affect the fluorescence of human Factor X, they decrease the cooperativity of the Ca2+ binding to the protein as judged by Hill plots of the Ca2+ -induced fluoresence quenching. EPR measurements indicate that there are three high affinity Mn2+ binding sites on human Factor X which can also bind Ca2+. Positive cooperativity was not observed for Mn2+ binding. These data indicate that Ca2+ can cause a conformational change of the Factor X molecule which allows the activation reaction to proceed. We propose that Mn2+ does not support the activation of human Factor X because it cannot induce a necessary conformational change in the absence of Ca2+.

Optimising filter life and performance efficiency in continuous renal replacement therapy has been a focus of considerable recent research. Larger high quality studies have predominantly focussed on optimal anticoagulation however CRRT is complex and filter life is also affected by vascular access, circuit and management factors. We performed a systematic search of the literature to identify and quantify the effect of vascular access, circuit and patient factors that affect filter life and presented the results as a meta-analysis. A systematic review and meta-analysis was performed by searching Pubmed (MEDLINE) and Ovid EMBASE libraries from inception to 29(th) February 2016 for all studies with a comparator or independent variable relating to CRRT circuits and reporting filter life. Included studies documented filter life in hours with a comparator other than anti-coagulation intervention. All studies comparing anticoagulation interventions were searched for regression or hazard models pertaining to other sources of variation in filter life. Eight hundred nineteen abstracts were identified of which 364 were selected for full text analysis. 24 presented data on patient modifiers of circuit life, 14 on vascular access modifiers and 34 on circuit related factors. Risk of bias was high and findings are hypothesis generating. Ranking of vascular access site by filter longevity favours: tunnelled semi-permanent catheters, femoral, internal jugular and subclavian last. There is inconsistency in the difference reported between femoral and jugular catheters. Amongst published literature, modality of CRRT consistently favoured continuous veno-venous haemodiafiltration (CVVHD-F) with an associated 44% lower failure rate compared to CVVH. There was a trend favouring higher blood flow rates. There is insufficient data to determine advantages of haemofilter membranes. Patient factors associated with a statistically significant worsening of filter life included mechanical

Background Generation of active procoagulant cofactor FVa and its subsequent association with the enzyme FXa to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Methods and Results Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied using plasma, whole blood and purified systems. Here we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B-domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. In line, tick feeding is impaired on TIX-5 immune rabbits displaying the in vivo importance of TIX-5. Conclusions Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. Based on our data we propose a revised blood coagulation scheme wherein direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors. PMID:23817575

The diagnosis of hemorrhagic shock in a patient with tachycardia and hypotension is generally straightforward. The difficulty lies in the selection of optimal approach for the patient. In this study on a canine model of deep hemorrhagic shock induced by bleeding, we used various amounts of fluid infusion to determine the most appropriate amount of fluid resuscitation. The study included 30 mongrel dogs. Mongrel dogs were randomized to three equal groups of 10. The control group received no treatment. The rapid infusion group received 60 mL/kg of lactated Ringer's solution in half an hour whereas the third group received 30 mL/kg during the same time period. The lactate decreased in the two treatment groups. The platelet and fibrinogen levels did not vary in the slow infusion group but decreased markedly in the rapid infusion group. In conclusion, fluid resuscitation should not be unnecessarily aggressive. The coagulation parameters are diluted in proportion with the amount of fluid given. Blood lactate level can be used as a guiding marker in evaluation of treatment effectiveness of hemorrhagic shock patients.

This study evaluated the effects of Al-coagulant sludge characteristics on the efficiency ofcoagulant recovery by acidification with H2SO4. Two sludge characteristics were studied: types of coagulant and textures of the suspended solid in raw water. The coagulant types are aluminium sulphate and polyaluminium chloride (PACl); the textures of the suspended solid are sand-based and clay-based. Efficiency of aluminium recovery at a pH of 2 was compared for different sludges obtained from water treatment plants in Taiwan. The results showed that efficiency of aluminium recovery from sludge containing clayey particles was higher than that from sludge containing sandy particles. As for the effect of coagulant types, the aluminium recovery efficiency for sludge using PACl ranged between 77% and 100%, whereas it ranged between 65% and 72% for sludge using aluminium sulphate as the coagulant. This means using PACl as the coagulant could result in higher recovery efficiency of coagulant and be beneficial for water treatment plants where renewable materials and waste reduction as the factors for making decisions regarding plant operations. However, other metals, such as manganese, could be released with aluminium during the acidification process and limit the use of the recovered coagulants. It is suggested that the recovered coagulants be used in wastewater treatment processes.

We conducted a retrospective study of 6,966 patients who had undergone tonsillectomy or adenotonsillectomy to evaluate the incidence and clinical features of previously unidentified coagulation disorders in patients who experienced postoperative hemorrhage (n = 201). We found that post-tonsillectomy hemorrhage secondary to unidentified coagulation disorders is extremely rare. However, normal coagulation values and an insignificant history do not rule out coagulation disorders. If diffuse, persistent, and bilateral bleeding is not related to arterial hypertension, dissection technique, or local infection, a rapid and detailed analysis of coagulationfactors should be considered.

The aim of this study was to determine the association between standard laboratory tests, coagulationfactor concentrations, and Rotation Thromboelastometry (ROTEM® delta, TEM® International GmbH, Munich, Germany) in patients undergoing major surgery with hemorrhage. In 45 patient's fibrinogen, factor VIII, factor XIII, International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), thrombin time, hemoglobin, leukocytes, and platelet count were simultaneously measured intraoperatively with ROTEM (EXTEM, INTEM, FIBTEM, APTEM) measurements. ROTEM parameters were: clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), and α-angle. Demographic and laboratory data were expressed as mean ± SD and median [range]; nonparametric Spearman rank correlations and multiple linear regressions were performed; P-values ≤0.003 were considered significant. Significant correlations (P ≤ 0.003) were found for CFT, α-angle, and MCF, in EXTEM, INTEM, and APTEM with platelets, INR, and fibrinogen. Factor VIII (18 measurements) showed a strong correlation (r ≥ 0.7 or r ≤ -0.7; all P ≤ 0.003) with MCF, CFT, and α-angle of EXTEM, INTEM, MCF of FIBTEM excluding CT of EXTEM, INTEM, FIBTEM and strong significant correlation for α-angle of APTEM and moderate for CFT and MCF of APTEM. A significant moderate to strong correlation of factor XIII with MCF of EXTEM, INTEM, FIBTEM, and APTEM was found. Hemoglobin was moderately correlated (r = 0.3-0.7 or r = -0.3 to -0.7) with MCF in APTEM (P = 0.003). A moderate to strong correlation of the standard coagulation tests with all ROTEM parameters was found, in particular the CT. The aPTT correlated significantly moderate to strong with CT, CFT, α-angle, and MCF of INTEM. However, multiple linear regressions were not able to show an influence of INR on ROTEM parameters except for APTEM-MCF. A significant impact of the aPTT on INTEM-CT was found. EXTEM, INTEM, and APTEM are significantly influenced

There is wide variation in the management of coagulation and blood transfusion practice in liver transplantation. The use of blood products intraoperatively is declining and transfusion free transplantations take place ever more frequently. Allogenic blood products have been shown to increase morbidity and mortality. Primary haemostasis, coagulation and fibrinolysis are altered by liver disease. This, combined with intraoperative disturbances of coagulation, increases the risk of bleeding. Meanwhile, the rebalancing of coagulation homeostasis can put patients at risk of hypercoagulability and thrombosis. The application of the principles of patient blood management to transplantation can reduce the risk of transfusion. This includes: preoperative recognition and treatment of anaemia, reduction of perioperative blood loss and the use of restrictive haemoglobin based transfusion triggers. The use of point of care coagulation monitoring using whole blood viscoelastic testing provides a picture of the complete coagulation process by which to guide and direct coagulation management. Pharmacological methods to reduce blood loss include the use of anti-fibrinolytic drugs to reduce fibrinolysis, and rarely, the use of recombinant factor VIIa. Factor concentrates are increasingly used; fibrinogen concentrates to improve clot strength and stability, and prothrombin complex concentrates to improve thrombin generation. Non-pharmacological methods to reduce blood loss include surgical utilisation of the piggyback technique and maintenance of a low central venous pressure. The use of intraoperative cell salvage and normovolaemic haemodilution reduces allogenic blood transfusion. Further research into methods of decreasing blood loss and alternatives to blood transfusion remains necessary to continue to improve outcomes after transplantation. PMID:24876736

The clotting process is a dynamic array of multiple processes which can be described in four phases: platelet plug initiation and formation, clotting process propagation by the coagulation cascade, clotting termination by antithrombotic mechanisms and clot removal by fibrinolysis. The liver plays a central role in each of these phases of clotting process, as it synthesizes the majority of coagulationfactors and proteins involved in fibrinolysis as well as thrombopoeitin, which is responsible for platelet production from megakaryocytes. Many pathological processes associated with cirrhosis, such as portal hypertension and endothelial dysfunction, as well as co-morbid conditions, may also alter the coagulation process. Consequently, patients with liver disease have a disturbed balance of procoagulant and anti-coagulantfactors which deviates from the normal coagulation cascade. This situation poses an additional problem in the diagnostic and therapeutic approach to this group of patients, since traditional coagulation test may not be reliable for assessing bleeding or thrombotic risk and traditional transfusional strategies may not be applicable in cirrhotic patients. In this article, we review the pathophysiological bases of coagulation abnormalities, in cirrhotic patients, the diagnostic therapeutic strategies to be followed and its impact on the clinical outcome in the cirrhotic patient.

There is wide variation in the management of coagulation and blood transfusion practice in liver transplantation. The use of blood products intraoperatively is declining and transfusion free transplantations take place ever more frequently. Allogenic blood products have been shown to increase morbidity and mortality. Primary haemostasis, coagulation and fibrinolysis are altered by liver disease. This, combined with intraoperative disturbances of coagulation, increases the risk of bleeding. Meanwhile, the rebalancing of coagulation homeostasis can put patients at risk of hypercoagulability and thrombosis. The application of the principles of patient blood management to transplantation can reduce the risk of transfusion. This includes: preoperative recognition and treatment of anaemia, reduction of perioperative blood loss and the use of restrictive haemoglobin based transfusion triggers. The use of point of care coagulation monitoring using whole blood viscoelastic testing provides a picture of the complete coagulation process by which to guide and direct coagulation management. Pharmacological methods to reduce blood loss include the use of anti-fibrinolytic drugs to reduce fibrinolysis, and rarely, the use of recombinant factor VIIa. Factor concentrates are increasingly used; fibrinogen concentrates to improve clot strength and stability, and prothrombin complex concentrates to improve thrombin generation. Non-pharmacological methods to reduce blood loss include surgical utilisation of the piggyback technique and maintenance of a low central venous pressure. The use of intraoperative cell salvage and normovolaemic haemodilution reduces allogenic blood transfusion. Further research into methods of decreasing blood loss and alternatives to blood transfusion remains necessary to continue to improve outcomes after transplantation.

The study of blood ex vivo can occur in closed or open systems, with or without flow. Microfluidic devices facilitate measurements of platelet function, coagulation biology, cellular biorheology, adhesion dynamics, pharmacology, and clinical diagnostics. An experimental session can accommodate 100s to 1000s of unique clotting events. Using microfluidics, thrombotic events can be studied on defined surfaces of biopolymers, matrix proteins, and tissue factor under constant flow rate or constant pressure drop conditions. Distinct shear rates can be created on a device with a single perfusion pump. Microfluidic devices facilitated the determination of intraluminal thrombus permeability and the discovery that platelet contractility can be activated by a sudden decrease in flow. Microfluidics are ideal for multicolor imaging of platelets, fibrin, and phosphatidylserine and provide a human blood analog to the mouse injury models. Overall, microfluidic advances offer many opportunities for research, drug testing under relevant hemodynamic conditions, and clinical diagnostics. PMID:23642241

Deletion of the α1,3-galactosyltransferase (GalT) gene in pigs has removed a major xenoantigen but has not eliminated the problem of dysregulated coagulation and vascular injury. Rejecting GalT knockout organ xenografts almost invariably show evidence of thrombosis and platelet sequestration, and primate recipients frequently develop consumptive coagulopathy. This review examines recent findings that illuminate potential mechanisms of this current barrier to successful xenotransplantation. The coagulation response to xenotransplantation differs depending on the type of organ and quite likely the distinct vasculatures. Renal xenografts appear more likely to initiate consumptive coagulopathy than cardiac xenografts, possibly reflecting differential transcriptional responses. Liver xenografts induce rapid and profound thrombocytopenia resulting in recipient death within days due to bleeding; ex-vivo data suggest that liver endothelial cells and hepatocytes are responsible for platelet consumption by a coagulation-independent process.It has been proposed that expression of recipient tissue factor on platelets and monocytes is an important trigger of consumptive coagulopathy. Finally, pigs transgenic for human anticoagulants and antithrombotics are slowly but surely coming on line, but have not yet been rigorously tested to date. Successful control of coagulation dysregulation in xenotransplantation may require different combinatorial pharmacological and genetic strategies for different organs.

Steady state kinetic studies have been performed to investigate the formation of thrombin from prothrombin by human coagulationFactor Xa in the presence of Ca2+ and phospholipid. The concentration of ligand which gives 50% of the maximum velocity (K0.5) is 2.3 mM for Ca2+, 7.4 microM for phospholipid, and 0.006 microM for prothrombin. Hill plots of the Ca2+ enhancement of the reaction give a Hill coefficient of 3.1, indicating positive cooperativity. The initial velocity patterns are consistent with an ordered addition of reactants with phospholipid as the second reactant to bind to the enzyme. Although our results do not differentiate between Ca2+ or the prothrombin substrate as the first reactant to bind to Factor Xa, it is established that Ca2+ can bind to Factor Xa in the absence of the other reactants. Thus, the most probable order of addition of reactants is Ca2+, phospholipid, and the prothrombin substrate. Plots of (v)-1 versus (prothrombin)-1 or (v)-1 versus [(Ca2+)3]-1 at several constant concentrations of phospholipid indicate that the major effect of phospholipid is to increase the turnover number of Factor Xa.

The development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigen-presenting cells, followed by endocytosis and presentation to naïve CD4+ T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIIIY1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIIIY1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo. In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro. Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity. PMID:27758819

The value of coagulationfactor V and VIII/V levels as prognostic indicators was assessed in 27 patients with fulminant hepatic failure and compared with other predictive indices. Admission factor V levels were significantly reduced in 22 patients with paracetamol induced hepatic failure compared with a healthy control group (median 9.5% v 103%, respectively; p less than 0.001) and with lower values in non-A non-B hepatitis (median 2.7%). Values in the seven patients who died after paracetamol overdose, considered together with the four who underwent liver transplantation (group median 5.1%), were significantly lower than in the 11 who survived (median 11.8%; p less than 0.01). Median admission factor VIII was higher in those who died or received a transplant than in those who survived (298% v 162%; p less than 0.05), with both results higher than in healthy volunteers (median 104%; p less than 0.01) but lower than in non-A non-B hepatitis (median 340%). The ratio of factor VIII/V on admission was less than 30 in all patients who survived paracetamol overdose (median 17) with corresponding values greater than 30 in 10 of 11 of those who died (median 39). A factor V result less than or equal to 10% on admission predicted an adverse outcome in 10 of 11 fatal cases, a 91% sensitivity which was greater than for the previously defined indicator of an arterial blood pH less than 7.30 on admission (sensitivity 82%). Prothrombin time at admission or on day 4 did not usefully predict outcome in our series. Predictive accuracy was 73% and 82% for factor V and admission acidosis respectively and 95% for factor V in conjunction with admission coma grade III or IV and factor VIII (ratio > 30). These criteria may be useful in selecting patients with paracetamol induced fulminant hepatic failure for transplantation. PMID:1740285

We test a hypothesis that the closed form of the C2 domain of coagulationfactor V is more stable than the open form in an aqueous environment using a two-dimensional free-energy calculation with a simple dielectric solvent model. Our result shows that while the free-energy difference between two forms is small, favoring the closed form, a two-dimensional free-energy surface (FES) reveals that a transition state (1.53 kcal/mol) exists between the two conformations. By mapping the one-dimensional order parameter DeltaQ onto the two-dimensional FES, we search the conformational change path with the highest Boltzmann weighting factor between the closed and open form of the factor V C2 domain. The predicted transition path from the closed to open form is not that of simple side chain movements, but instead concerted movements of several loops. We also present a one-dimensional free-energy profile using a collective order parameter, which in a coarse manner locates the energy barriers found on the two-dimensional FES.

Measurement of coagulationfactor activity using absolute physico-chemical techniques is not possible and estimation therefore relies on comparative bioassay relative to a reference standard with a known or assigned potency. However the inherent variability of locally prepared and calibrated reference standards can give rise to poor agreement between laboratories and methods. Harmonisation of measurement between laboratories at the international level relies on the availability of a common source of calibration for local reference standards and this is provided by the World Health Organization (WHO) International Standards which define the International Unit for the analyte. This article describes the principles, practices and problems of biological standardisation and the development and use of reference standards for assays of coagulationfactors, with particular emphasis on WHO International Standards for both concentrates and plasma. Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.

Description is given of changes in blood coagulability found in diseases of the rabbit, including malignant tumor, spontaneous infections, non-bacterial diseases and lesions, and hemorrhagic states specifically induced. The changes involved variously the time of onset of blood coagulation, clot formation rate and the rate and extent of clot retraction. PMID:19869243

Alteration in activity of blood coagulationfactors in dogs with acute hepatic injury caused by oral carbon tetrachloride dosing was studied. CoagulationFactors II, VII and IX were dramatically reduced within 48 hours but recovered to normal in the next five days. Because surgery is rarely performed on dogs with hepatic necrosis, the use of fresh whole blood tranfusion to improve the coagulation defect in hepatic injury was also studied. Transfusion was found to have only a temporary beneficial effect. PMID:4253461

The intimate connection between coagulation and inflammation in the pathogenesis of vascular disease has moved more and more into focus of clinical research. This paper focuses on the essential components of this interplay in the settings of cardiovascular disease and acute coronary syndrome. Tissue factor, the main initiator of the extrinsic coagulation pathway, plays a central role via causing a proinflammatory response through activation of coagulationfactors and thereby initiating coagulation and downstream cellular signalling pathways. Regarding activated clotting factors II, X, and VII, protease-activated receptors provide the molecular link between coagulation and inflammation. Hereby, PAR-1 displays deleterious as well as beneficial properties. Unravelling these interrelations may help developing new strategies to ameliorate the detrimental reciprocal aggravation of inflammation and coagulation. PMID:22518344

A recombinant single-chain variable antibody fragment (scFv) KM33 was previously described as a ligand that can inhibit the function of blood coagulationfactor VIII (FVIII). This scFv was previously derived from an individual with anti-FVIII antibodies manifested in FVIII functional deficiency (Hemophilia A) and expressed in bacteria. In the present work, we describe an alternative approach for fast and easy production of KM33 in insect cells (Spodoptera frugiperda). The KM33 gene was codon-optimized and expressed in secreted form using a baculovirus system. The protein was isolated using metal-affinity and size-exclusion chromatography to purity of about 96% and yield of 0.4-1.2 mg per 120 mL of culture, based on several independent expression experiments. In a binding assay using surface plasmon resonance, the insect cell-derived KM33 (iKM33) was qualified as a high-affinity ligand for FVIII. Epitope specificity of iKM33 on FVIII (C1 domain) was confirmed by testing the binding with a relevant mutant of FVIII. In several FVIII functional tests (factor Xa generation, APTT clotting, thrombin generation and video microscopy clot growth assays), iKM33 strongly inhibited FVIII activity in accordance with the clinical effect of the parental antibody. Therefore, the expressed protein was concluded to be fully functional and applicable in various assays with FVIII. Published by Elsevier Inc.

A large fraction of factor VIII in blood originates from liver sinusoidal endothelial cells although extrahepatic sources also contribute to plasma factor VIII levels. Identification of cell-types other than endothelial cells with the capacity to synthesize and release factor VIII will be helpful for therapeutic approaches in hemophilia A. Recent cell therapy and bone marrow transplantation studies indicated that Küpffer cells, monocytes and mesenchymal stromal cells could synthesize factor VIII in sufficient amount to ameliorate the bleeding phenotype in hemophilic mice. To further establish the role of blood cells in expressing factor VIII, we studied various types of mouse and human hematopoietic cells. We identified factor VIII in cells isolated from peripheral and cord blood, as well as bone marrow. Co-staining for cell type-specific markers verified that factor VIII was expressed in monocytes, macrophages and megakaryocytes. We additionally verified that factor VIII was expressed in liver sinusoidal endothelial cells and endothelial cells elsewhere, e.g., in the spleen, lungs and kidneys. Factor VIII was well expressed in sinusoidal endothelial cells and Küpffer cells isolated from human liver, whereas by comparison isolated human hepatocytes expressed factor VIII at very low levels. After transplantation of CD34(+) human cord blood cells into NOD/SCIDγNull-hemophilia A mice, fluorescence activated cell sorting of peripheral blood showed >40% donor cells engrafted in the majority of mice. In these animals, plasma factor VIII activity 12 weeks after cell transplantation was up to 5% and nine of 12 mice survived after a tail clip-assay. In conclusion, hematopoietic cells, in addition to endothelial cells, express and secrete factor VIII: this information should offer further opportunities for understanding mechanisms of factor VIII synthesis and replenishment.

Chitosan is a potential substitute for traditional aluminium salts in water treatment systems. This study compared the characteristics of humic acid (HA) removal by using acid-soluble chitosan, water-soluble chitosan, and coagulant mixtures of chitosan with aluminium sulphate (alum) or polyaluminium chloride (PACl). In addition, we evaluated their respective coagulation efficiencies at various coagulant concentrations, pH values, turbidities, and hardness levels. Furthermore, we determined the size and settling velocity of flocs formed by these coagulants to identify the major factors affecting HA coagulation. The coagulation efficiency of acid- and water-soluble chitosan for 15 mg/l of HA was 74.4% and 87.5%, respectively. The optimal coagulation range of water-soluble chitosan (9-20 mg/l) was broader than that of acid-soluble chitosan (4-8 mg/l). Notably, acid-soluble chitosan/PACl and water-soluble chitosan/alum coagulant mixtures exhibited a higher coagulation efficiency for HA than for PACl or alum alone. Furthermore, these coagulant mixtures yielded an acceptable floc settling velocity and savings in both installation and operational expenses. Based on these results, we confidently assert that coagulant mixtures with a 1:1 mass ratio of acid-soluble chitosan/PACl and water-soluble chitosan/alum provide a substantially more cost-effective alternative to using chitosan alone for removing HA from water.

The blood coagulation system of 66 consecutive patients undergoing consecutive liver transplantations was monitored by thrombelastograph and analytic coagulation profile. A poor preoperative coagulation state, decrease in levels of coagulationfactors, progressive fibrinolysis, and whole blood clot lysis were observed during the preanhepatic and anhepatic stages of surgery. A further general decrease in coagulationfactors and platelets, activation of fibrinolysis, and abrupt decrease in levels of factors V and VIII occurred before and with reperfusion of the homograft. Recovery of blood coagulability began 30–60 min after reperfusion of the graft liver, and coagulability had returned toward baseline values 2 hr after reperfusion. A positive correlation was shown between the variables of thrombelastography and those of the coagulation profile. Thrombelastography was shown to be a reliable and rapid monitoring system. Its use was associated with a 33% reduction of blood and fluid infusion volume, whereas blood coagulability was maintained without an increase in the number of blood product donors. PMID:3896028

We study coagulation in the flow field of a time-periodic deterministic chaotic flow and focus on the simplest case: point particles convected without diffusion and allowed to coagulate with probability 1 when the distance is less than d. An analysis of the underlying physics is presented. Under ''well-mixed'' conditions the system behaves as if the particles were moved by Brownian motion, and a simple kinetic model describes the main results. The poorly mixed case is considerably more complex. Spatial inhomogeneities result from competition between the rate of coagulation and mixing, and trapping and leaking of clusters due to Kolmogorov-Arnold-Moser surfaces.

Coagulation is an important mechanism in the growth of interstellar and interplanetary dust particles. The microphysics of the coagulation process was theoretically analyzed as a function of the physical properties of the coagulating grains, i.e., their size, relative velocities, temperature, elastic properties, and the van der Waal interaction. Numerical calculations of collisions between linear chains provide the wave energy in individual particles and the spectrum of the mechanical vibrations set up in colliding particles. Sticking probabilities are then calculated using simple estimates for elastic deformation energies and for the attenuation of the wave energy due to absorption and scattering processes.

Chemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulationfactor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity. PMID:27791187

Chemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulationfactor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.

The inhibitory effect on the activated coagulationfactor X activity (anti-Xa activity) in plasma and urine of danaparoid sodium (DAS, CAS 9005-49-6) was compared with that of dalteparin sodium (DLS, CAS 9041-08-1) and heparin (CAS 9005-49-6) after single intravenous administration at a dose of 640 anti-Xa U/kg to male rabbits. The elimination of half-life of DAS was 9.90 h and was 6.0 times longer than that of DLS and 16.5 times longer than that of heparin. The area under the plasma concentration-time curve (AUC) of DAS was 47.13 +/- 14.55 anti-Xa U.h/ml and was 2.4 times larger than that of DLS and 2.9 times larger than that of heparin. The urinary cumulative excretion of anti-Xa activity of DAS and DLS was 42.6 +/- 6.4% and 16.4 +/- 0.8% of dose, respectively, in 24 h after dosing, respectively. But the anti-Xa activity in urine was not detected at any sampling points after administration of heparin. DAS has a longer elimination half-life and a higher renal excretion of anti-Xa activity than that of DLS and heparin. Therefore, in comparison to DLS and heparin, it seems that the anticoagulant activity of DAS has a long duration.

The genus Citrus of the family Rutaceae includes many species e.g. Citrus indica, Citrus aurantifolia and Citrus limon, among which Citrus limon L. Burm. f. has been reported to have highest antimicrobial activity. It is used as antidote against certain venom, due to its platelet inhibitory effect and also reported to have hypocholesterolemic effect. However its anticoagulant and thrombolytic effect were not been investigated, hence a prospective in-vitro/in-vivo study was designed to determine the effect of Citrus limon on blood parameters, coagulation and anticoagulation factors. In-vitro tests revealed highly significant increase in thrombin time and activated partial thromboplastin time by Citrus limon, whereas fibrinogen concentration was significantly reduced in comparison to control, however prothrombin time was not affected significantly. In-vivo testing of Citrus limon was done at three different doses i.e. 0.2ml/kg, 0.4ml/kg and 0.6ml/kg in healthy rabbits. Significant changes were observed in hematological parameters such as erythrocytes, hemoglobin and mean corpuscular hemoglobin concentration. Bleeding time and thrombin time was significantly prolonged and there was increase in protein C and thrombin antithrombin complex levels. These results may be due to inactivation of thrombin because it significantly decreases fibrinogen concentration and inhibit platelet aggregation. Citrus limon showed maximal anticoagulant effect at 0.4ml/kg, which suggest that Citrus limon possesses an anti-thrombin component and could prevent thrombosis playing a cardio protective role.

The first case report describes an extremely prolonged activated partial thromboplastin time (APTT) in a patient with no history of increased bleeding tendency. Heparin use was excluded. The APTT mixing study combined with the medical history suggests a deficiency in one of the non-essential coagulationfactors. This was confirmed by factor XII activity of <1%. The second case report describes a prolonged APTT in a patient with no history of increased bleeding tendency. The negative bleeding tendency in combination with a failure of the mixing study to correct the coagulation assay results suggests a factor inhibitor, most probably lupus anticoagulant. Indeed, the lupus anticoagulant was positive and the anti-cardiolipin antibody titre was also positive. Aberrations in the process of haemostasis can be efficiently screened using a platelet count, an APTT, a PT and a thorough physical examination combined with a thorough medical history taking. Common causes of prolonged PT and/or APTT are the use of oral anticoagulants or heparin, vitamin K deficiency and liver disease. Other causes include coagulationfactor deficiencies, coagulationfactor inhibitors and diffuse intravascular coagulation.

Background VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) – the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulationfactor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1. Results In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme. Conclusions The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism. PMID:24491178

A survey is given on the peculiar characteristics of laboratory methods for analyzing the blood coagulation system with special regard to the preanalytical, analytical and postanalytical phase. Routinely used methods are described.

Brain cephaline-based reagents for evaluating activated partial thromboplastin time (APTT) and soybean phosphatides with ellagic acid complex activator with intermediate metal ions have been studied. The sensitivity of these reagents to internal clotting factors (VIII and IX) and heparin is determined by phospholipid nature and type of metal. The results help obtain highly active and sensitive APTT reagents.

Pathological coagulation parameters may reflect life-threatening hemorrhagic or thromboembolic diseases but may also be a laboratory result without any clinical significance, result from in vitro phenomena or preanalytical errors. This article gives an overview of potential pitfalls in coagulation diagnostics, lists the differential diagnoses of pathological coagulation parameters and describes further steps in the diagnostic approach to clarify pathological results. The focus lies on coagulation parameters that are frequently determined in routine clinical investigations, e.g. platelet count, prothrombin time, activated partial thromboplastin time (aPTT) and fibrinogen. Besides heparin, fondaparinux, danaparoid, and vitamin K antagonists, direct factor Xa inhibitors and direct thrombin inhibitors are nowadays available for therapeutic anticoagulation. This article gives an overview of the influence of anticoagulants on coagulation parameters which depends on the dose, the time of the last administration, as well as the method used for the determination of coagulation parameters. Moreover, common reasons for elevation of the fibrin degradation product D-dimer are presented. The clinical utility of D-dimer assays is limited by their poor specificity. Elevated D-dimer concentrations can be found in various diseases and also under normal physiological circumstances (e.g. in the elderly). Thus, the most useful clinical application of D-dimer is evidence of normal values to essentially rule out venous thromboembolism.

Diallyl trisulfide (DAT)-rich garlic oil was fed to Sprague-Dawley rats and the effects of this DAT-rich garlic oil on bleeding time, clotting time and anticoagulation factors were examined. Garlic oil supplement at 5 or 50mg garlic oil/kg bodyweight significantly prolonged bleeding time and thrombin time, and enhanced anticoagulation factor activity, such as antithrombin III and protein C (P<0.05). These results suggested that the anticoagulant action of DAT-rich garlic oil was due to inhibition and/or inactivation of thrombin. In addition, DAT-rich garlic oil benefits blood anticoagulation factors, which might further prevent the development of thrombus formation. However, the intake of garlic oil at high dose significantly increased plasma fibrinogen concentration (P<0.05), and affected the levels of several hematological parameters such as erythrocyte count, hemoglobin and platelets (P<0.05). The adverse effect of high doses of garlic oil might further influence the hemostatic balance. Therefore, the concentration of DAT-rich garlic oil should be carefully considered in its application. Supplementation of garlic oil at 5mg/kg bodyweight has anticoagulation effect in this animal study.

Whole blood Coagulation analyzers (WBCAs) are widely used point-of-care (POC) testing devices found primarily in cardiothoracic surgical suites and cardia catheterization laboratories. Most of these devices can perform a number of coagulation tests that provide information about a patient's blood clotting status. Clinicians use the results of the WBCA tests, which are available minutes after applying a blood sample, primarily to monitor the effectiveness of heparin therapy--an anticoagulation therapy used during cardiopulmonary bypass (CPB) surgery, angioplasty, hemodialysis, and other clinical procedures. In this study we evaluated five WBCAs from four suppliers. Our testing focused on the applications for which WBCAs are primarily used: Monitoring moderate to high heparin levels, as would be required, for example, during CPB are angioplasty. For this function, WCBAs are typically used to perform an activated clotting time (ACT) test or, as one supplier refers to its test, a heparin management test (HMT). All models included in this study offered an ACT test or an HMT. Monitoring low heparin levels, as would be required, for example,during hemodialysis. For this function, WBCAs would normally be used to perform either a low-range ACT (LACT) test or a whole blood activated partial thromboplastin time (WBAPTT) test. Most of the evaluated units could perform at least one of these tests; one unit did not offer either test and was therefore not rated for this application. We rated and ranked each evaluated model separately for each of these two applications. In addition, we provided a combined rating and ranking that considers the units' appropriateness for performing both application. We based our conclusions on a unit's performance and humans factor design, as determined by our testing, and on its five-year life-cycle cost, as determined by our net present value (NPV) analysis. While we rated all evaluated units acceptable for each appropriate category, we did

Prothrombin time (PT) is widely used as the monitor of oral anticoagulant therapy using ISI/NR system which WHO recommended. However, a main clinical usefulness of an Owren-type combined reagent (TT) also remains in the monitoring of warfarin in Europe and Japan. Recently, a TT reagent utilizing a recombinant bovine tissue factor (TF) expressed by silkworm-baculovirus system has been developed. The purpose of this study is to investigate the sensitivity to coagulationfactors (FII, FV, FVII, FX) of PT (rabbit brain and recombinant human) and TT (bovine brain and recombinant bovine) in INR, and the correlation among those reagents using 53 plasma samples from patients treated with warfarin. The INR results were calculated using a certified INR calibrator, "AK-CALIBRANT", according to the method of "Direct" INR determination. The sensitivity to FII, FVII and FX of those reagents results similar behavior in INR, but the sensitivity to FV of PT reagents were generally higher than that of TT reagents. The correlation coefficient between recombinant PT and recombinant TT was 0.979. There were a good agreement between two TT reagents (bovine brain and recombinant bovine) in INR (r = 0.998). It was apparent that a variance between PT and TT was dependent on the sensitivity to FV level during the course of warfarin treatment in a clinical case. In conclusion, PT and TT reagents gave generally acceptable correlation in INR, and our results indicate that the Owren-type TT reagent is also well suited for monitoring warfarin using local INR calibration according to WHO recommendation.

A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulationfactor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P< .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274.

A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulationfactor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P < .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274. PMID:26755710

Background Evidence for the associations of single nucleotide polymorphisms (SNPs) in the F7 gene and factor VII (FVII) levels and with risk of coronary heart disease (CHD) is inconsistent. We examined whether F7 tagging SNPs and haplotypes were associated with FVII levels, coagulation activation markers (CAMs) and CHD risk in two cohorts of UK men. Methods Genotypes for eight SNPs and baseline levels of FVIIc, FVIIag, and CAMs (including FVIIa) were determined in 2773 healthy men from the Second Northwick Park Heart Study (NPHS-II). A second cohort, Whitehall II study (WH-II, n=4055), was used for replication analysis of FVIIc levels and CHD-risk. Results In NPHS-II the minor alleles of three SNPs (rs555212, rs762635, and rs510317; haplotype H2) were associated with higher levels of FVIIag, FVIIc, and FVIIa, while the minor allele for two SNPs (I/D323, and rs6046; haplotype H5) were associated with lower levels. Adjusted for classical risk factors, H2 carriers had a CHD Hazard Ratio of 1.34 (CI 95%: 1.12–1.59; independent of FVIIc), while H5 carriers had a CHD-risk of 1.29 (CI 95%: 1.01–1.56; not independent of FVIIc) and significantly lower CAMs. Effects of haplotypes on FVIIc levels were replicated in WH-II, as was association of H5 with higher CHD-risk (pooled-estimate OR 1.16 [1.00–1.36], P=0.05), but surprisingly, H2 exhibited a reduced risk for CHD. Conclusion tSNPs in the F7 gene strongly influence FVII levels. The haplotype associated with low FVIIc level, with particularly reduced functional activity, was consistently associated with increased risk for CHD, while the haplotype associated with high FVIIc level was not. PMID:20735728

Specific mass spectrometry and direct activated factor X (Xa)- and thrombin inhibition assays do not allow determination of the reversal of anticoagulant effects of non-vitamin K direct oral anticoagulants (NOACs) by prothrombin complex concentrate (PCC). The objective of this study was the evaluation of the applicability of a variety of commercially available global coagulation assays in analyzing the reversal of NOAC anticoagulation by PCC. Plasma and whole blood were spiked with apixaban or dabigatran and PCC was added to these samples. Prothrombin time (PT), modified PT (mPT), activated partial prothrombin time (APTT), thrombography (CAT method) and thromboelastography (ROTEM, TEG) were performed. Assays triggered by contact activation (APTT, INTEM) did not show inhibitor reversal by PCC. Assays triggered by tissue factor (TF) showed NOAC type and NOAC concentration dependent anticoagulation reversal effects of PCC ranging from partial normalization to overcorrection of the following parameters: clotting or reaction time (PT, mPT TEG-TF, EXTEM, FIBTEM); angle in thromboelastography (TEG-TF); thrombin generation (CAT) lag time, endogenous thrombin potential (ETP) and peak thrombin. Extent of reversal was assay reagent dependent. ETP (5 pM TF) was the only parameter showing complete reversal of anticoagulation by PCC for all NOACs ranging from 200 to 800 μg/L. ETP fits with the concept that reversal assessment of NOAC anticoagulation by PCC should be based on measurements on the clotting potential or thrombin generating potential of the plasma or whole blood patient sample. Low sensitivity of ETP for NOACs and its correlation with bleeding are issues that remain to be resolved.

Blood flow regulates coagulation and fibrin formation by controlling the transport, or mass transfer, of zymogens, co-factors, enzymes, and inhibitors to, from, and within a growing thrombus. The rate of mass transfer of these solutes relative to their consumption or production by coagulation reactions determines, in part, the rate of thrombin generation, fibrin deposition, and thrombi growth. Experimental studies on the influence of blood flow on specific coagulation reactions are reviewed here, along with a theoretical framework that predicts how flow influences surface-bound coagulation binding and enzymatic reactions. These flow-mediated transport mechanisms are also used to interpret the role of binding site densities and injury size on initiating coagulation and fibrin deposition. The importance of transport of coagulation proteins within the interstitial spaces of thrombi is shown to influence thrombi architecture, growth, and arrest. PMID:27133256

Coagulation is an important process in hemostasis and comprises a complicated interaction of multiple enzymes and proteins. We have developed a mechanistic quantitative model of the coagulation network. The model accurately describes the time courses of coagulationfactors following in vivo activation as well as in vitro blood coagulation tests of prothrombin time (PT, often reported as international normalized ratio (INR)) and activated partial thromboplastin time (aPTT). The model predicts the concentration-time and time-effect profiles of warfarin, heparins, and vitamin K in humans. The model can be applied to predict the time courses of coagulation kinetics in clinical situations (e.g., hemophilia) and for biomarker identification during drug development. The model developed in this study is the first quantitative description of the comprehensive coagulation network.

Purpose The aim of this study was to investigate prophylactic treatment effects in Korean patients with severe hemophilia A. Methods A prospective study of 32 severe hemophilia A patients was conducted with the approval of the Institutional Review Board at the Eulji University Hospital. Two patients received primary prophylaxis; whereas, the other 30 patients were divided into 2 groups-secondary prophylaxis (n=15) and on-demand (n=15)-on the basis of their consent for secondary prophylaxis. A 20-25 IU/kg dose of factor VIII concentrate was administered to the primary and secondary prophylaxis group patients every 3 days for 1 year. The prophylactic effect was evaluated by observing changes in the Pettersson scores, annual number of total and joint bleeds, and factor VIII consumption for 1 year. Results No moderate or severe bleeding was observed, and the Pettersson scores remained unchanged during the prophylaxis period in the patients who received primary prophylactic treatment. After the treatment was changed from on-demand to secondary prophylaxis, the annual number of total and joint bleeds in the secondary prophylaxis group decreased by 64.4%±13.0% and 70.0%±15.2%, respectively. The average increase in Pettersson scores within 1 year was 0.5±0.8 and 1.3±1.1 in the secondary prophylaxis and on-demand groups, respectively. Prophylactic effects were also observed in patients >17 years who had nearly the same initial Pettersson scores. Conclusion Intermediate-dose prophylactic treatment may delay hemarthropathy progression and prevent its occurrence in Korean severe hemophilia A patients. PMID:23908669

The binding of coagulationfactor X (FX) to the hexon of adenovirus (Ad) 5 is pivotal for hepatocyte transduction. However, vectors based on Ad35, a subspecies B Ad, are in development for cancer gene therapy, as Ad35 utilizes CD46 (which is upregulated in many cancers) for transduction. We investigated whether interaction of Ad35 with FX influenced vector tropism using Ad5, Ad35, and Ad5/Ad35 chimeras: Ad5/fiber(f)35, Ad5/penton(p)35/f35, and Ad35/f5. Surface plasmon resonance (SPR) revealed that Ad35 and Ad35/f5 bound FX with approximately tenfold lower affinities than Ad5 hexon-containing viruses, and electron cryomicroscopy (cryo-EM) demonstrated a direct Ad35 hexon:FX interaction. The presence of physiological levels of FX significantly inhibited transduction of vectors containing Ad35 fibers (Ad5/f35, Ad5/p35/f35, and Ad35) in CD46-positive cells. Vectors were intravenously administered to CD46 transgenic mice in the presence and absence of FX-binding protein (X-bp), resulting in reduced liver accumulation for all vectors. Moreover, Ad5/f35 and Ad5/p35/f35 efficiently accumulated in the lung, whereas Ad5 demonstrated poor lung targeting. Additionally, X-bp significantly reduced lung genome accumulation for Ad5/f35 and Ad5/p35/f35, whereas Ad35 was significantly enhanced. In summary, vectors based on the full Ad35 serotype will be useful vectors for selective gene transfer via CD46 due to a weaker FX interaction compared to Ad5.

Few randomized studies have reported on the use of factor IX (FIX) for secondary prophylaxis in haemophilia B patients. This study aimed to evaluate the efficacy and safety of two secondary prophylaxis regimens of recombinant coagulation FIX, nonacog alfa, compared with on-demand therapy. Male subjects aged 6-65 years with severe or moderately severe haemophilia B (FIX:C ≤ 2, n = 50) and ≥12 bleeding episodes (including ≥6 haemarthroses episodes) within 12 months of study participation were enrolled in this multicentre, randomized, open-label, four-period crossover trial. The primary measure was the annualized bleeding rate (ABR) of two prophylactic regimens vs. on-demand therapy. In the intent-to-treat group, mean ABR values were 35.1, 2.6 and 4.6 for the first on-demand period, the 50 IU kg(-1) twice-weekly period, and the 100 IU kg(-1) once-weekly period respectively. Differences in ABR between the first on-demand period and both prophylaxis regimens were significant (P

Lymphoceles are among the most common postoperative complications of pelvic lymphadenectomy (PL), with a reported incidence of 1% to 50%. Symptoms are pelvic pain, leg edema, gastrointestinal obstruction, obstructive uropathy, and deep vein thrombosis, and severe complications such as sepsis and lymphatic fistula formation. After laparoscopic PL, we tested the prevention of lymphoceles using collagen patch coated with the human coagulationfactors (TachoSil, Nycomed International Management GmbH, Zurich, Switzerland) on 55 patients with endometrial cancer stages IB to II who had undergone laparoscopy. The authors divided the patients into 2 laparoscopy groups: PL plus TachoSil (group 1: 26 patients) and PL without TachoSil in a control group (group 2: 29 patients), as historical cohort of patients who underwent PL between 2010 and 2012. We collected surgical parameters, and the patients underwent ultrasound examination on postoperative days 7, 14, and 28. The main outcome measures were the development of symptomatic or asymptomatic lymphoceles, the need for further surgical intervention, as adverse effect of surgery, and the drainage volume and duration. The same number of lymph nodes in both groups was removed; group 1 showed a lower drainage volume. Lymphoceles developed in 5 patients in group 1 and in 15 patients in group 2; of these, only 2 patients were symptomatic in group 1 and 5 patients were symptomatic in group 2, without statistical difference and no percutaneous drainage request. In this preliminary investigation, the intraoperative laparoscopy application of TachoSil seems to reduce the rate of postoperative lymphoceles after PL, providing a useful additional treatment option for reducing drainage volume and preventing lymphocele development after PL.

A recombinant fusion protein linking coagulationfactor IX (FIX) with human albumin (rIX-FP) has been developed to facilitate hemophilia B treatment by less frequent FIX dosing. This first-in-human dose-escalation trial in 25 previously treated subjects with hemophilia B (FIX ≤ 2 IU/dL) examined the safety and pharmacokinetics of 25, 50, and 75 IU/kg rIX-FP. Patients in the 50-IU/kg cohort underwent a comparative pharmacokinetics assessment with their previous FIX product (plasma-derived or recombinant). No allergic reactions or inhibitors were observed. Four mild, possibly treatment-related adverse events were reported. In the 50-IU/kg cohort (13 subjects), the mean half-life of rIX-FP was 92 hours, more than 5 times longer than the subjects' previous FIX product. After 25 or 50 IU/kg rIX-FP administration, the baseline-corrected mean FIX activity remained elevated at day 7 (7.4 IU/dL and 13.4 IU/dL, respectively) and day 14 (2.5 IU/dL and 5.5 IU/dL, respectively). The incremental recovery of rIX-FP was higher than both recombinant and plasma-derived FIX (1.4 vs 0.95 and 1.1 IU/dL per IU/kg, respectively). These results demonstrated both the safety and improved pharmacokinetics of rIX-FP, thus indicating this new product with extended half-life as possibly able to control and prevent bleeding with less frequent injection. The trial was registered at www.clinicaltrials.gov as no. NCT01233440. PMID:22859609

features of serum proteome. The signature included upregulation of factors involved in acute or inflammatory response but also downregulation of plasma apolipoproteins and factors involved in blood coagulation.

This study aims to explore the relation between childhood obesity and procoagulant and anticoagulant systems. Fifty-one obese children and 32 normal-weighted children with similar age and gender distribution and between ages of 5 and 16 years were recruited to the study. Antropometric measures of all subjects, existence of any accompanying disease, and medication histories had been recorded. Full blood count, procoagulant, and anticoagulant coagulation tests were run for all subjects. When hematologic variables of obese children were compared with those of healthy controls, it was found that average erythrocyte hemoglobin concentration, erythrocyte distribution width, and platelet count of obese children are significantly higher than healthy control group. It was also found that fibrinogen, thrombin time, factor (F) VIII, FIX, FX, and von Willebrand factor levels of obese children are higher than healthy control group. By contrast, antithrombin levels of obese children are found to be lower. In our study, we found that there is a procoagulant increase in the coagulation system activity of obese children compared to non-obese healthy children, whereas there is a significant decrease in anticoagulant system. These changes occurred in obese patients, especially higher levels of plasma procoagulant factors such as fibrinogen, FVIII, FIX, and von Willebrand factor, lead us to think that there is an activity in these patients at endothelial level. Further studies are needed on endothelial activity of obese children.

Potable water treatment facilities may become an important barrier in limiting human exposure to engineered nanoparticles (ENPs) as ENPs begin to contaminate natural aquatic systems. Coagulation of ENPs will likely be a major process that controls the ENP fate and the subsequent removal in the aqueous phase. The influence that source water quality has on ENP coagulation is still relatively unknown. The current study uses a 2(3) x 2(4-1) fractional factorial design to identify seven key surface water constituents that affect multiwall carbon nanotube (MWCNT) coagulation. These seven factors include: influent concentrations of kaolin, organic matter (OM), alginate, and MWCNTs; type and dosage of coagulant; and method of MWCNT stabilization. MWCNT removal was most affected by coagulant type and dosage, with alum outperforming ferric chloride at circumneutral pH. None of the other factors were universally significant but instead depended on coagulant type, dose, and method of stabilization. In all cases where factors were found to have a significant impact on MWCNT removal, however, the relationship was consistent: higher influent concentrations of kaolin and alginate improved MWCNT removal while higher influent concentrations of OM hindered MWCNT coagulation. Once MWCNTs are released into the natural environment, their coagulation behavior will be determined by the type and quantity of pollutants (i.e., factors) present in the aquatic environment and are governed by the same mechanisms that influence the colloidal stability of "natural" nanoparticles.

Rheological measurements in biological liquids yield insights into homeostasis and provide information on important molecular processes that affect fluidity. We present a fully automated cantilever-based method for highly precise and sensitive measurements of microliter sample volumes of human blood plasma coagulation (0.009 cP for viscosity range 0.5-3 cP and 0.0012 g/cm(3) for density range 0.9-1.1 g/cm(3)). Microcantilever arrays are driven by a piezoelectric element, and resonance frequencies and quality factors of sensors that change over time are evaluated. A highly accurate approximation of the hydrodynamic function is introduced that correlates resonance frequency and quality factor of cantilever beams immersed in a fluid to the viscosity and density of that fluid. The theoretical model was validated using glycerol reference solutions. We present a surface functionalization protocol that allows minimization of unspecific protein adsorption onto cantilevers. Adsorption leads to measurement distortions and incorrect estimation of the fluid parameters (viscosity and density). Two hydrophilic terminated self-assembled monolayers (SAMs) sensor surfaces are compared to a hydrophobic terminated SAM coating. As expected, the hydrophobic modified surfaces induced the highest mass adsorption and could promote conformational changes of the proteins and subsequent abnormal biological activity. Finally, the activated partial thromboplastin time (aPTT) coagulation assay was performed, and the viscosity, density, and coagulation rate of human blood plasma were measured along with the standard coagulation time. The method could extend and improve current coagulation testing.

A marked abnormality of the intrinsic coagulation system was observed in a patient with melanomatosis and frank melanuria. With successful treatment of the tumour there was a parallel improvement in the clotting abnormality. This defect was shown to be a deficiency of factor XI together with a previously unrecognised factor. This is distinct from either Fletcher or Fitzgerald factors and appears to act between factors XI and X in the coagulation sequence. PMID:874111

To determine the role of activated status of peroxisome proliferator-activated receptorγ/nuclear factor-ΚB (PPAR-γ/NF-ΚB ) in coagulation disorders induced by sepsis. Forty male Sprague-Dawley (SD) rats were randomly divided into four groups, n=10 in each group: control group, lipopolysaccharide (LPS) challenged group, rosiglitazone (ROSI, selective agonist of PPAR-γ) pretreatment group, and GW9662 (PPAR-γ antagonist) pretreatment group. The sepsis model was reproduced by injection of 6 mg/kg LPS via sublingual vein, and the rats in control group were injected with 2 mL/kg normal saline. The rats in ROSI pretreatment group were given 0.3 mg/kg ROSI by sublingual venous injection followed by injection of LPS 30 minutes later; and in GW9662 pretreatment group rats were given 0.3 mg/kg GW9662 by sublingual venous injection followed by 0.3 mg/kg ROSI 15 minutes later, followed by injection of LPS 30 minutes later. Blood was collected at 4 hours after LPS administration, and the expressions of PPAR-γ and NF-ΚBp65 in peripheral blood mononuclear cell (PBMC) were determined with immunocytocheminal technique and graph analysis. Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and D-dimer were determined simultaneously. (1) PPAR-γ/NF-ΚB pathway: the expressions of PPAR-γ and NF-ΚBp65 were lowered in control group, and they were expressed in cytoplasm. In LPS challenged group the expression of PPAR-γ (gray value) was slightly increased but with no significant difference as compared with control group (111.01±4.06 vs. 98.46±5.99, P>0.05). In ROSI pretreatment group the expression of PPAR-γ (gray value) was significantly higher than that in LPS challenged group (214.38±5.79 vs. 111.01±4.06, P<0.01), with dislocation into nuclei. In GW9662 pretreatment group the expression of PPAR-γ (gray value) was lowered but without significant difference compared with that of control group (44.21±2.64 vs. 98.46±5.99, P>0

Management of coagulation in neurosurgical procedures is challenging. In this contest, it is imperative to avoid further intracranial bleeding. Perioperative bleeding can be associated with a number of factors, including anticoagulant drugs and coagulation status but is also linked to the characteristic and the site of the intracranial disorder. The aim of this review will be to focus primarily on the new evidence regarding the management of coagulation in patients undergoing craniotomy for neurosurgical procedures. Antihemostatic and anticoagulant drugs have shown to be associated with perioperative bleeding. On the other hand, an increased risk of venous thromboembolism and hypercoagulative state after elective and emergency neurosurgery, in particular after brain tumor surgery, has been described in several patients. To balance the risk between thrombosis and bleeding, it is important to be familiar with the perioperative changes in coagulation and with the recent management guidelines for anticoagulated patients undergoing neurosurgical procedures, in particular for those taking new direct anticoagulants. We have considered the current clinical trials and literature regarding both safety and efficacy of deep venous thrombosis prophylaxis in the neurosurgical population. These were mainly trials concerning both elective surgical and intensive care patients with a poor grade intracranial bleed or multiple traumas with an associated severe traumatic brain injury (TBI). Coagulation management remains a major issue in patients undergoing neurosurgical procedures. However, in this field of research, literature quality is poor and further studies are necessary to identify the best strategies to minimize risks in this group of patients.

Coagulation and condensation/evaporation combined with atmospheric dispersion are the main processes responsible for the evolution of aerosol particle size distributions and number concentrations emitted from localized sources. A crucial question is: what fraction of freshly emitted particles survive intra-coagulation effect to persist in the atmosphere and become available for further interaction with background aerosols?. The difficulty in estimating this quantity, designated as the number survival fraction, arises due chiefly to the joint action of atmospheric diffusion with nonlinear coagulation effects which are computationally intensive to handle. We provide a simplified approach to evaluate this quantity in the context of instantaneous (puff) and continuous (plume) releases based on a reduction of the respective coagulation-diffusion equations under the assumption of a constant coagulation kernel ( K). The condensation/evaporation processes, being number conserving, are not included in the study. The approach consists of constructing moment equations for the evolution of number concentration and variance of the spatial extension of puff or plume in terms of either time or downstream distance. The puff model, applicable to instantaneous releases is solved within a 3-D, spherically symmetric framework, under an additional assumption of a constant diffusion coefficient ( D) which renders itself amenable to a closed form solution that provides a benchmark for developing the solution to the plume model. The latter case, corresponding to continuous releases, is discussed within a 2-D framework under the assumptions of constant advection velocity ( U) and space dependent diffusion coefficient expressed in terms of turbulent energy dissipation rate ( ɛ). The study brings out the special effect of the coagulation-induced flattening of the spatial concentration profiles because of which particle sizes will be larger at the centre of a Gaussian puff. For a puff of

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by coagulantfactor VIII (FVIII) deficiency. The conventional treatment involves the administration of recombinant human FVIII (rhFVIII) preparations. In this study, the mammary gland ‘bioreactor’ is designed to specifically and efficiently express a foreign protein hFVIII in the mammary glands of transgenic mice. We constructed a P1A3-hFVIIIBD vector directed by the mammary gland-specific P1A3 promoter, and transiently transfected HC11 cells and mouse mammary glands with P1A3-hFVIIIBD or CMV-hFVIIIBD vectors directed by a ubiquitous cytomegalovirus (CMV) promoter, respectively. We also generated P1A3-hFVIIIBD and CMV-hFVIIIBD transgenic mice by microinjection, respectively. Our data indicated that both vectors effectively expressed hFVIIIBD in HC11 cells at the transcription level, and hFVIIIBD protein was efficiently expressed in mouse milk after the injection of the hFVIIIBD vectors into mouse mammary glands during lactation. In both CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice, hFVIIIBD proteins were efficiently expressed in the mammary glands at the mRNA and protein levels. No significant difference was observed in hFVIIIBD levels between the CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice (P > 0.05). However, the activity of hFVIII in CMV-directed transgenic mice was slightly higher than that in P1A3-directed transgenic mice (P

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by coagulantfactor VIII (FVIII) deficiency. The conventional treatment involves the administration of recombinant human FVIII (rhFVIII) preparations. In this study, the mammary gland 'bioreactor' is designed to specifically and efficiently express a foreign protein hFVIII in the mammary glands of transgenic mice. We constructed a P1A3-hFVIIIBD vector directed by the mammary gland-specific P1A3 promoter, and transiently transfected HC11 cells and mouse mammary glands with P1A3-hFVIIIBD or CMV-hFVIIIBD vectors directed by a ubiquitous cytomegalovirus (CMV) promoter, respectively. We also generated P1A3-hFVIIIBD and CMV-hFVIIIBD transgenic mice by microinjection, respectively. Our data indicated that both vectors effectively expressed hFVIIIBD in HC11 cells at the transcription level, and hFVIIIBD protein was efficiently expressed in mouse milk after the injection of the hFVIIIBD vectors into mouse mammary glands during lactation. In both CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice, hFVIIIBD proteins were efficiently expressed in the mammary glands at the mRNA and protein levels. No significant difference was observed in hFVIIIBD levels between the CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice (P > 0.05). However, the activity of hFVIII in CMV-directed transgenic mice was slightly higher than that in P1A3-directed transgenic mice (P < 0.05). While hFVIIIBD was present in multiple organs in CMV-hFVIIIBD mice, P1A3-hFVIIIBD mice showed negligible hFVIIIBD expression in organs other than the mammary glands. This study demonstrated that the mammary gland-specific P1A3-hFVIIIBD vector was more suitable for the generation of hFVIIIBD mammary gland bioreactor.

People are advised to reduce their intake of saturated fat and replace it by carbohydrate to avoid coronary heart disease. It is unknown whether sucrose and starchy foods, two major sources of carbohydrates, have similar effects on cardiovascular risk markers if incorporated as a replacement for saturated fat into diets eaten ad libitum. We served 20 healthy, normal-weight women aged 21 to 52 years three strictly controlled diets ad libitum: FAT, high in total fat (46% of total energy [E%]) and saturated fat (21 E%); STARCH, high in total carbohydrates (59 E%) and low in sucrose (2.5 E%); and SUCROSE, high in total carbohydrates (59 E%) and sucrose (23.2 E%). The diets were eaten in randomized order for a period of 2 weeks. Blood lipids, factor VII coagulant activity (FVIIc), and fibrinogen concentrations were measured with subjects in the fasted state (9:45 AM) and the postabsorptive state (6:00 PM). STARCH was associated with lower total cholesterol (mean difference, 0.34 mmol/L; 95% confidence interval [CI], 0.18 to 0.50), low-density lipoprotein (LDL) cholesterol (0.25 mmol/L; 95% CI, 0.13 to 0.37), fasting triglycerides (0.15 mmol/L; 95% CI, 0.07 to 0.23), nonfasting triglycerides (0.44 mmol/L; 95% CI, 0.30 to 0.58), and nonfasting FVIIc (9.8%; 95% CI, 3.8 to 15.8) than SUCROSE. Compared with FAT, STARCH resulted in a desirable decrease of LDL cholesterol and nonfasting FVIIc. STARCH was also associated with a minor weight loss (0.7 kg) that was not found on the other 2 diets. We conclude that starchy foods with a natural content of dietary fiber can be recommended as substitutes for saturated fat in the dietary prevention of coronary heart disease. According to the present short-term findings in healthy females, substitution with sucrose is not advisable.

An investigation of dye decolorization from synthetic dye solutions using the non-ionic, water-soluble, high molecular weight seed gums Ipomoea dasysperma and guar gum as coagulants was undertaken. The use of galactomannans derived from plants in this system presents a sustainable method of textile effluent treatment. These natural coagulants extracted from plants proved to be workable alternatives to conventional coagulants like polyaluminum chloride, as they are biodegradable, safe to human health, are cost effective when compared to imported chemicals and have a wider effective dosage range for flocculation of various colloidal suspensions. Coagulant dose and coagulation pH are important factors influencing the mechanism of coagulation. Also the type and chemical structure of the dye plays an important role in the coagulation process. The seed gums alone were found to be effective for decolorization of direct dye and in combination with PAC their coagulation efficiency was well extended even for reactive and acid dyes.

Coagulation process is widely used for removal of natural organic matters (NOM) and for water color intensity reduction. The efficiency of coagulation process depends on many different factors. Aim of this research is to investigate coagulation process under different conditions. During the research coagulation process was held at different pH values (5.5; 6.0; 6.5), at different water alkalinity and at different water turbidity. It was found that removal of NOM and water color intensity reduction is most effective at pH values from 5.5 to 6.0. At these conditions water color intensity reduction is most efficient, but removal of dissolved organic carbon (DOC) is the lowest. During the research it was also found that different water alkalinity and turbidity do not make significant influence on efficiency of coagulation process.

Asthma is a chronic airway disease characterized by paroxysmal airflow obstruction evoked by irritative stimuli on a background of allergic lung inflammation. Currently, there is no cure for asthma, only symptomatic treatment. In recent years, our understanding of the involvement of coagulation and anticoagulant pathways, the fibrinolytic system, and platelets in the pathophysiology of asthma has increased considerably. Asthma is associated with a procoagulant state in the bronchoalveolar space, further aggravated by impaired local activities of the anticoagulant protein C system and fibrinolysis. Protease-activated receptors have been implicated as the molecular link between coagulation and allergic inflammation in asthma. This review summarizes current knowledge of the impact of the disturbed hemostatic balance in the lungs on asthma severity and manifestations and identifies new possible targets for asthma treatment.

The liver plays a key role in hemostasis as the site of synthesis of many of the proteins involved in the coagulation, antithrombotic and fibrinolytic systems that interact to both establish hemostasis, and preventing thrombosis. The common laboratory tests, prothrombin time (PT) and activated partial thromboplastin time (aPTT), evolved from studies of plasma clotting in test tubes. Such studies laid the basis for the coagulation cascade model of hemostasis. However, thought has evolved to place a greater emphasis on the active roles of cells in localizing and regulating hemostasis. The PT and aPTT do not reflect the roles of cellular elements in hemostasis, nor do they reflect the crucial roles of antithrombotic and fibrinolytic systems. Thus, though the PT may indeed reflect the synthetic capacity of the liver, it does not accurately reflect the risk of bleeding or thrombosis in patients with liver failure.

Argon Plasma Coagulation (APC) is an application of gas discharges in argon in electrosurgery, which is increasingly used especially in endoscopy. The major application fields are haemostasis, tissue devitalization and tissue reduction. This review describes the physics and technology of electrosurgery and APC. Some characteristics of the argon discharge are shown and discussed, and thermal effects in biological tissue are described. Subsequently, examples of medical applications are given. PMID:20204117

Allogeneic mesenchymal stem cells (MSCs) show great potential for the treatment of military and civilian trauma, based on their reduced immunogenicity and ability to modulate inflammation and immune function in the recipient. Although generally considered to be safe, MSCs express tissue factor (TF), a potent activator of coagulation. In the current study, we evaluated multiple MSC populations for tissue factor expression and pro-coagulant activity in order to characterize safety considerations for systemic use of MSCs in trauma patients who may have altered coagulation homeostasis. Multiple MSC populations derived from either human adipose tissue or bone marrow were expanded in the recommended stem cell media. Stem cell identity was confirmed using a well-characterized panel of positive and negative markers. Tissue factor expression on the cell surface was evaluated by flow cytometry with anti-CD142 antibody. Effects on blood coagulation were determined by thromboelastography (TEG) and calibrated automated thrombogram (CAT) assays using platelet poor plasma or whole blood. MSCs express tissue factor on their surfaces and are pro-coagulant in the presence of blood or plasma. The adipose-derived MSCs (Ad-MSC) evaluated were more pro-coagulant and expressed more tissue factor than bone marrow MSCs (BM-MSCs), which showed a greater variability in TF expression. BM-MSCs were identified that exhibited low pro-coagulant activity, whereas all Ad-MSCs examined exhibited high pro-coagulant activity. The percentage of cells in a given population expressing surface tissue factor correlates roughly with functional pro-coagulant activity. MSC tissue factor expression and pro-coagulant activity change over time in culture. All MSC populations are not equivalent; care should be taken to select cells for clinical use that minimize potential safety problems and maximize chance of patient benefit. Adipose-derived MSCs appear more consistently pro-coagulant than BM-MSCs, presenting a

Crystals of habu coagulationfactor IX-binding protein have been obtained at pH 6.5 and 4.6 and characterized by X-ray diffraction. Coagulationfactor IX-binding protein isolated from Trimeresurus flavoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca{sup 2+}-binding site with differing affinity (K{sub d} values of 14 and 130 µM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the affinity of the low-affinity site was reduced considerably. In order to identify which site has high affinity and also to investigate the Ca{sup 2+}-releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29 Å resolution, respectively; the former crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 60.7, b = 63.5, c = 66.9 Å, β = 117.0°, while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8 Å, β = 110.4°.

Disseminated intravascular coagulation (DIC) is a syndrome characterized by systemic intravascular activation of coagulation, leading to a widespread deposition of fibrin in the circulation. There is ample experimental and pathological evidence that the fibrin deposition contributes to multiple organ failure. The massive and ongoing activation of coagulation may result in depletion of platelets and coagulationfactors, which may cause bleeding (consumption coagulopathy). The syndrome of DIC is well known in the medical literature for centuries, although a more precise description of the underlying mechanisms had to await the 20th century. Initial ideas on a role of the contact activation system as the primary trigger for the systemic activation of coagulation as well as a presumed hyperfibrinolytic response in DIC have been found to be misconceptions. Experimental and clinical evidence now indicate that the initiation of coagulation in DIC is caused by tissue factor expression, which in combination with downregulated physiological anticoagulant pathways and impaired fibrinolysis leads to widespread fibrin deposition. In addition, an extensive bidirectional interaction between coagulation and inflammation may further contribute to the pathogenesis of DIC.

This study evaluated the removal of naturally occurring arsenic in a full-scale (106-mgd) conventional treatment plant. When the source water was treated with 3--10 mg/L of ferric chloride or 6, 10, or 20 mg/L of alum, arsenic removal was 81--96% (ferric chloride) and 23--71% (alum). Metal concentrations in the sludge produced during this study were below the state`s current hazardous waste levels at all coagulant dosages. No operational difficulties were encountered.

We retrospectively analyzed 60 cases of pediatric patients who received allogeneic stem cell transplantation (SCT) between 2000 and 2008, using the tentative scoring system for evaluation of early (<30 days) coagulation disorders. In the 41 patients who survived, D-dimer levels showed a transient increase 2 weeks after SCT and normalized thereafter, but these levels were persistently elevated in the 19 patients who died. Of 19 patients with a positive score, 11 died of transplantation-related complications [transplantation-related mortality (TRM) = 0.579] within 1 year, while none of the 41 with a negative score died during the same period. Since 2009, 12 of 30 patients had positive scores within 30 days after SCT. Intervention with recombinant human thrombomodulin (rhTM) was introduced for patients with a positive score, and 10 of these patients survived (TRM = 0.167) along with a dramatic improvement of D-dimer level. Although the effects of this treatment were observed in a limited number of patients, our observations suggest that early coagulation disorder after allogeneic SCT is a strong prognostic factor for TRM, and that intervention with rhTM improves TRM.

Blood coagulation often accompanies bacterial infections and sepsis and is generally accepted as a consequence of immune responses. Though many bacterial species can directly activate individual coagulationfactors, they have not been shown to directly initiate the coagulation cascade that precedes clot formation. Here we demonstrated, using microfluidics and surface patterning, that the spatial localization of bacteria substantially affects coagulation of human and mouse blood and plasma. Bacillus cereus and Bacillus anthracis, the anthrax-causing pathogen, directly initiated coagulation of blood in minutes when bacterial cells were clustered. Coagulation of human blood by B. anthracis required secreted zinc metalloprotease InhA1, which activated prothrombin and factor X directly (not via factor XII or tissue factor pathways). We refer to this mechanism as 'quorum acting' to distinguish it from quorum sensing--it does not require a change in gene expression, it can be rapid and it can be independent of bacterium-to-bacterium communication.

The recent increase in soymilk and tofu (coagulated soymilk) consumption, especially in Western countries, is due to the recognition of the health benefits of soy foods; consumption of soybean would prevent heart diseases. Since the amount and the type of coagulated biomolecules (such as isoflavones) will vary with the type of coagulant, this will inevitably alter their biological activity. This study sought to assess the effect of some coagulants (calcium chloride, alum, and steep water from pap production) commonly used in the production of tofu in Nigeria on the serum cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels in albino rats fed tofu for 14 days. The results of this study revealed that there was a significant difference (P < .05) in the total phenol [calcium chloride (0.9%), alum (0.8%), and steep water (1.2%)] content of all the tofu produced. Furthermore, feeding albino rats with tofu and water ad libitum for 14 days caused a significant decrease (P < .05) in serum cholesterol and LDL when compared with the control. Conversely, there was a significant increase (P < .05) in serum HDL when compared with the control. However, rats fed steep water-coagulated tofu had the lowest serum levels of cholesterol and LDL, followed by those fed alum- and calcium chloride-coagulated tofu, respectively, while those fed with alum-coagulated tofu had the highest serum HDL level, closely followed by those fed steep water-coagulated tofu. It was therefore concluded that of all the coagulants, steep water appeared to be the most promising coagulant with regard to the production of tofu with a high hypocholesterolemic effect based on the low serum cholesterol and LDL levels and high HDL level.

Coagulation-flocculation process is one of the most commonly used treatment process in water and wastewater treatment. Particles (PA) and phosphates (P) removal are the main objectives in wastewater coagulation. There is a general agreement on the dominant mechanism of PA and P removal during coagulation. While it is agreed that the PA and P removal reactions are competitive and takes place simultaneously, there is no clear understanding on the ratio of distribution of coagulants among the PA and P removal. The ratio can be significantly influenced by the content of PA and P, in addition to other water and coagulant quality factors. This paper attempts to provide a qualitative ratio of coagulant distribution based on PA:P proportion in raw water and OH:Al ratio in coagulants.

The detection of blood coagulation is very important in therapeutics and surgery. It is necessary to determine the overall time taken for blood clotting, production rate of thrombin, presence or absence of blood coagulationfactors, etc. In this paper a new method for detection of blood coagulation is presented. This method is based on the fiber-optic sensor and allows for the study of different ways of blood clotting (such as blood coagulation and platelets aggregation) separately, thus enhancing the precision of determination. The method for determining the blood coagulation presented possesses high precision in monitoring the process of coagulation. An elaborate mathematical model of the process of blood coagulation has been developed to help the computer handle obtained data.

Nanoparticle interactions with the blood coagulation system can be beneficial or adverse depending on the intended use of a nanomaterial. Nanoparticles can be engineered to be procoagulant or to carry coagulation-initiating factors to treat certain disorders. Likewise, they can be designed to be anticoagulant or to carry anticoagulant drugs to intervene in other pathological conditions in which coagulation is a concern. An overview of the coagulation system was given and a discussion of a desirable interface between this system and engineered nanomaterials was assessed in part I, which was published in the May 2013 issue of Nanomedicine. Unwanted pro- and anti-coagulant properties of nanoparticles represent significant concerns in the field of nanomedicine, and often hamper the development and transition into the clinic of many promising engineered nanocarriers. This part will focus on the undesirable effects of engineered nanomaterials on the blood coagulation system. We will discuss the relationship between the physicochemical properties of nanoparticles (e.g., size, charge and hydrophobicity) that determine their negative effects on the blood coagulation system in order to understand how manipulation of these properties can help to overcome unwanted side effects.

Nanoparticle interactions with the blood coagulation system can be beneficial or adverse depending on the intended use of a nanomaterial. Nanoparticles can be engineered to be procoagulant or to carry coagulation-initiating factors to treat certain disorders. Likewise, they can be designed to be anticoagulant or to carry anticoagulant drugs to intervene in other pathological conditions in which coagulation is a concern. An overview of the coagulation system was given and a discussion of a desirable interface between this system and engineered nanomaterials was assessed in part I, which was published in the May 2013 issue of Nanomedicine. Unwanted pro- and anti-coagulant properties of nanoparticles represent significant concerns in the field of nanomedicine, and often hamper the development and transition into the clinic of many promising engineered nanocarriers. This part will focus on the undesirable effects of engineered nanomaterials on the blood coagulation system. We will discuss the relationship between the physicochemical properties of nanoparticles (e.g., size, charge and hydrophobicity) that determine their negative effects on the blood coagulation system in order to understand how manipulation of these properties can help to overcome unwanted side effects. PMID:23730696

Measurement of blood viscoelasticity during clotting provides a direct metric of haemostatic conditions. Therefore, technologies that quantify blood viscoelasticity at the point-of-care are invaluable for diagnosing coagulopathies. We present a new approach, Optical Thromboelastography (OTEG) that measures the viscoelastic properties of coagulating blood by evaluating temporal laser speckle fluctuations, reflected from a few blood drops. During coagulation, platelet-fibrin clot formation restricts the mean square displacements (MSD) of scatterers and decelerates speckle fluctuations. Cross-correlation analysis of speckle frames provides the speckle intensity temporal autocorrelation, g2(t), from which MSD is deduced and the viscoelastic modulus of blood is estimated. Our results demonstrate a close correspondence between blood viscoelasticity evaluated by OTEG and mechanical rheometry. Spatio-temporal speckle analyses yield 2-dimensional maps of clot viscoelasticity, enabling the identification of micro-clot formation at distinct rates in normal and coagulopathic specimens. These findings confirm the unique capability of OTEG for the rapid evaluation of patients’ coagulation status and highlight the potential for point-of-care use. Spatial maps of blood viscoelasticity index, G, during clotting obtained from a normal patient (top row) and a hypo-coagulable patient with low levels of clotting factors (bottom row) at 0, 1, 14, and 30 minutes after kaolin activation. Micro-clots of significant G values appear at early times (~1 min) and continue to progress to form a large blood clot over 30 min in the normal patient. In contrast, in the hypo-coagulable sample, micro-clots of comparable G are only visible at 14 min and the extent and overall clot strength is considerably reduced compared to the normal patient even at 30 min. Scale bars are 100 μm long. These results demonstrate the high sensitivity and spatial resolution of OTEG for detecting incipient micro

Smoluchowski's equation for rapid coagulation is used to describe the kinetics of gelation, in which the coagulation kernel K/sub i/j models the bonding mechanism. For different classes of kernels we derive criteria for the occurrences of gelation, and obtain critical exponents in the pre- and postgelation stage in terms of the model parameters; we calculate bounds on the time of gelation t/sub c/, and give an exact postgelation solution for the model K/sub i/j = (ij)/sup ..omega../ (..omega..>1/2) and K/sub i/j = ..cap alpha../sup i/+j (..cap alpha..>1). For the model K/sub i/j = i/sup ..omega../+j/sup ..omega../ (..omega..<1, without gelation) initial solutions are given. It is argued that the kernel K/sub i/japprox. (ij)/sup ..omega../ with ..omega..approx. =1-1/d (d is dimensionality) effectively models the sol-gel transformation is polymerizing systems and approximately accounts for the effects of cross-linking and steric hindrance neglected in the classical theory of Flory and Stockmayer (..omega.. = 1). For all ..omega.. the exponents, tau = ..omega..+3/2 and sigma = ..omega..-1/2, ..gamma.. = (3/2-..omega..)/(..omega..-1/2) and ..beta.. = 1, characterize the size distribution, at the slightly below the gel point, under the assumption that scaling is valid.

The lack of comprehensive simulation models for wastewater coagulation is one of the obstacles to achieving optimal coagulant dosing. Two approaches for developing a model to describe the coagulation process are presented. The comprehensiveness in describing the influent quality with several parameters, rather than with one parameter, is identified to give high efficiency in dosing models based on algorithms constructed using the partial least squares method. The concept was tested on two full-scale wastewater treatment plants, with coagulant savings and effluent quality improvements. Significant differences were observed with the increase of online parameters in the models. The second approach is based on distribution of coagulant for particle and phosphate removal processes and is discussed for inert fractions. The concept is integrated into existing simulation software as a module. The calibration results and dosage predictions are demonstrated.

Pharmacogenetic research has shown that genetic variation may influence statin responsiveness. Statins exert a variety of beneficial effects beyond lipid lowering, including antithrombotic effects, which contribute to the risk reduction of cardiovascular disease. Statins have been shown to influence the expression of coagulationfactors II, V, VII, XII and XIII. Data from a large randomized clinical trial of pravastatin, designed to show efficacy relative to usual care, were used to investigate whether a pharmacogenetic effect of polymorphisms in genes coding for coagulationfactors II, V, VII, XII and XIII is associated with reduced fatal coronary heart disease (CHD) and nonfatal myocardial infarction, combined CHD and all-cause mortality. The Genetics of Hypertension Associated Treatment is an ancillary study of the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial. The genotyped population in the lipid-lowering trial of Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial included 9624 participants randomly assigned to pravastatin or to usual care. The efficacy of pravastatin in reducing risk of all-cause mortality, CHD and nonfatal myocardial infarction and combined CHD, was compared among genotype strata by examining an interaction term in a proportional hazards model. None of the polymorphisms were associated with the clinical outcomes. For the F7 (-323) ins/del polymorphism there was no interaction with pravastatin for either outcome. For both the F5 Arg506Gln G>A (rs6025) polymorphism and F7 Arg353Gln G>A (rs6046) polymorphism there were no interactions with pravastatin in relation to all-cause mortality, but there were significant interactions with combined CHD [interaction hazard ratio = 1.33, 95% confidence interval (1.01-1.76) and interaction hazard ratio = 1.92, 95% confidence interval (1.00-3.65), respectively]. There were no interactions between the polymorphisms in the other coagulation genes and

Background Pharmacogenetic research has shown that genetic variation may influence statin responsiveness. Statins exert a variety of beneficial effects beyond lipid lowering, including antithrombotic effects, which contribute to the risk reduction of cardiovascular disease. Statins have been shown to influence the expression of coagulationfactors II, V, VII, XII and XIII. AimData from a large randomized clinical trial of pravastatin, designed to show efficacy relative to usual care, were used to investigate whether a pharmacogenetic effect of polymorphisms in genes coding for coagulationfactors II, V, VII, XII and XIII is associated with reduced fatal coronary heart disease (CHD) and nonfatal myocardial infarction, combined CHD and all-cause mortality. Methods The Genetics of Hypertension Associated Treatment is an ancillary study of the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial. The genotyped population in the lipid-lowering trial of Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial included 9624 participants randomly assigned to pravastatin or to usual care. The efficacy of pravastatin in reducing risk of all-cause mortality, CHD plus nonfatal myocardial infarction and combined CHD, was compared among genotype strata by examining an interaction term in a proportional hazards modelAQ2. Results None of the polymorphisms were associated with the clinical outcomes. For the F7 (−323) del/ins polymorphism there was no interaction with pravastatin for either outcome. For both the F5 Arg506Gln G>A (rs6025) polymorphism and F7 Arg353Gln G>A (rs6046) polymorphism there were no interactions with pravastatin in relation to all-cause mortality, but there were significant interactions with combined CHD [interaction hazard ratioλ=λ1.33, 95% confidence interval (1.01−1.76) and interaction hazard ratioλ=λ1.92, 95% confidence interval (1.00−3.65), respectively]AQ3. There were no interactions between the

The role of coagulation disorders in the pathogenesis of (recurrent) ischemic stroke is uncertain. Therefore, the clinical utility of screening patients with ischemic stroke for these conditions and the therapeutic implications of a detected coagulation disorder in a patient who experienced ischemic stroke are uncertain. We reviewed the currently available data on the relationship between various inherited and acquired coagulation abnormalities (factor V Leiden and prothrombin G20210A mutations, deficiencies of protein C, protein S and anti-thrombin, hyperhomocysteinemia, the antiphospholipid syndrome and increased levels of fibrinogen) and ischemic stroke. Based on the existing evidence we discuss the usefulness of screening stroke patients for prothrombotic conditions and current recommendations regarding the optimal management of ischemic stroke patients in whom a coagulation disorder is found.

The move to a low-carbon economy has generated renewed interest in microalgae for the production of biofuels with the potential mutual benefit of wastewater treatment. However, harvesting has been identified as a limiting factor to the economic viability of this process. This paper explores the harvesting of microalgae using high-pressure gas without the addition of coagulants. Coagulation of microalgae under high-pressure gas was found to be an efficient method to separate algae from suspension. The critical coagulation pressures (CCPs) for H(2) and CO(2) were determined to be 6.1 and 6.2 MPa, respectively. The CO(2)-induced decrease in solution pH positively influenced coagulation rates, without appearing to affect the CCP. This approach could be beneficial for the economic removal of microalgae from solution for the production of both biofuels and biomedical compounds without the addition of non-environmentally friendly chemicals.

Disseminated intravascular coagulation (DIC) is a syndrome characterized by systemic intravascular activation of coagulation, leading to widespread deposition of fibrin in the circulation. Recent knowledge on important pathogenetic mechanisms that may lead to DIC has resulted in novel preventive and therapeutic approaches to patients with DIC. The diagnosis of DIC can be made by sensitive laboratory tests; however, most of these tests are not readily available in a clinical setting. A reliable diagnosis can also be made on the basis of a small series of laboratory tests that can be combined in a scoring algorithm. The cornerstone of the management of DIC is the specific and vigorous treatment of the underlying disorder. Strategies aimed at the inhibition of coagulation activation may theoretically be justified and have been found beneficial in experimental and clinical studies. These strategies comprise inhibition of tissue factor-mediated activation of coagulation or restoration of physiological anticoagulant pathways.

A theory of aerosol coagulation rates resulting from continuum-regime Brownian coagulation in the presence of size-dependent particle thermophoresis is developed and explored here. We are motivated by a wide variety of applications in which particle Brownian coagulation occurs in a nonisothermal gas where differential thermophoretic drift contributes to, but does not dominate, the encounter frequency between suspended spherical particles (e.g., mist droplets) of different sizes. We employ a Smoluchowski-like population-balance to demonstrate the relative roles of Brownian diffusion and thermophoresis in shaping the short and long time (asymptotic or “coagulation-aged”) mist-droplet size distribution (DSD) function. To carry out these combined-mechanism DSD-evolution calculations we developed a rational “coupled” coagulation rate constant (allowing for simultaneous Brownian diffusion and relative thermophoretic drift) rather than simply adding the relevant individual coagulation “kernels.” Dimensionless criteria are provided to facilitate precluding other coagulation mechanisms not considered here (such as simultaneous sedimentation or Marangoni-flow-induced mist-droplet phoresis) and potential complications not included in the present model [as finite-rate coalescence, initial departures from the continuum (Stokes drag-) limit, and even dense (nonideal) vapor effects].

A theory of aerosol coagulation rates resulting from continuum-regime brownian coagulation in the presence of size-dependent particle thermophoresis is developed and explored here. We are motivated by a wide variety of applications in which particle brownian coagulation occurs in a nonisothermal gas where differential thermophoretic drift contributes to, but does not dominate, the encounter frequency between suspended spherical particles (e.g., mist droplets) of different sizes. We employ a Smoluchowski-like population-balance to demonstrate the relative roles of brownian diffusion and thermophoresis in shaping the short and long time (asymptotic or "coagulation-aged") mist-droplet size distribution (DSD) function. To carry out these combined-mechanism DSD-evolution calculations we developed a rational "coupled" coagulation rate constant (allowing for simultaneous brownian diffusion and relative thermophoretic drift) rather than simply adding the relevant individual coagulation "kernels." Dimensionless criteria are provided to facilitate precluding other coagulation mechanisms not considered here (such as simultaneous sedimentation or Marangoni-flow-induced mist-droplet phoresis) and potential complications not included in the present model [as finite-rate coalescence, initial departures from the continuum (Stokes drag-) limit, and even dense (nonideal) vapor effects].

Thymoquinone (THQ) is a major component of black seeds. Given that both THQ and black seeds exhibit anti-cancer and anti-inflammatory activities, we hypothesized that THQ will affect cancer-associated thrombosis (CAT), which is primarily triggered by tissue factor (TF) and inflammation. The effect of both black seed-extracted and purchased (“pure”) THQ on normal blood coagulation was tested with in vitro thromboelastography (TEG) and activated partial thromboplastin time (aPTT) coagulation assays. The effect of pure THQ on CAT was tested with aPTT assay using pancreatic cancer cell lines that are either positive or negative for TF, and with TEG assay using lipopolysaccharide as an inflammatory trigger. Additionally, the direct effect of THQ on the inactivation of factors IIa and Xa was assessed. Since TNF-α facilitates crosstalk between inflammation and thrombosis by triggering the NF-κB pathway, we tested THQ’s ability to interfere with this communication with a luciferase assay. Both extracted and pure THQ had minimal effects on normal blood coagulation. Pure THQ reversed CAT initiated by both TF and inflammation to basal levels (p < 0.001). Mechanistically, while THQ had minimal to no effect on factor IIa and Xa inactivation, it strongly reduced the effects of TNF-α on NF-κB elements (p < 0.001). THQ has a minimal effect on basal coagulation and can reverse CAT in vitro, possibly by interfering with the crosstalk between inflammation and coagulation. This study suggests the utility of THQ as a preventative anticoagulant and/or as a supplement to existing chemotherapies and anticoagulant therapies. PMID:27043539

Thymoquinone (THQ) is a major component of black seeds. Given that both THQ and black seeds exhibit anti-cancer and anti-inflammatory activities, we hypothesized that THQ will affect cancer-associated thrombosis (CAT), which is primarily triggered by tissue factor (TF) and inflammation. The effect of both black seed-extracted and purchased ("pure") THQ on normal blood coagulation was tested with in vitro thromboelastography (TEG) and activated partial thromboplastin time (aPTT) coagulation assays. The effect of pure THQ on CAT was tested with aPTT assay using pancreatic cancer cell lines that are either positive or negative for TF, and with TEG assay using lipopolysaccharide as an inflammatory trigger. Additionally, the direct effect of THQ on the inactivation of factors IIa and Xa was assessed. Since TNF-α facilitates crosstalk between inflammation and thrombosis by triggering the NF-κB pathway, we tested THQ's ability to interfere with this communication with a luciferase assay. Both extracted and pure THQ had minimal effects on normal blood coagulation. Pure THQ reversed CAT initiated by both TF and inflammation to basal levels (p < 0.001). Mechanistically, while THQ had minimal to no effect on factor IIa and Xa inactivation, it strongly reduced the effects of TNF-α on NF-κB elements (p < 0.001). THQ has a minimal effect on basal coagulation and can reverse CAT in vitro, possibly by interfering with the crosstalk between inflammation and coagulation. This study suggests the utility of THQ as a preventative anticoagulant and/or as a supplement to existing chemotherapies and anticoagulant therapies.

Single nucleotide polymorphism (SNP) genotyping methods are widely used for the detection of hereditary thrombophilias caused by genetic defects in the coagulation system. The hereditary thrombophilias are frequently associated with higher incidences of point mutations in hemostasis (F2 20210G>A, F5 1691G>A) and folate metabolism (MTHFR 677C>Т, MTHFR 1298A>C) genes. Moreover, the combination of gene abnormalities in F2 or/and MTHFR with F5 Leiden mutation leads to increased risk of developing thrombosis. Thus, simultaneous detection of the multiple gene mutations in a sample has important clinical relevance. The microchip-based multiplex real time PCR for estimation of allele specific polymorphism in hemostatic and folate metabolism genes presented here has a high efficiency and may be used for laboratory diagnosis. The optimized protocol for estimation of 4 different types of genetic polymorphisms allowed PCR to be performed with minimal quantity of DNA template and PCR reagents including Taq polymerase and a short-term thermocycling.

The structure and metabolism of HDL are linked to their major apolipoproteins (apo) A-I and A-II. HDL metabolism is very dynamic and depends on the constant remodeling by lipases, lipid transfer proteins and receptors. HDL exert several cardioprotective effects, through their antioxidant and antiinflammatory capacities and through the stimulation of reverse cholesterol transport from extrahepatic tissues to the liver for excretion into bile. HDL also serve as plasma reservoir for C and E apolipoproteins, as transport vehicles for a great variety of proteins, and may have more physiological functions than previously recognized. In this review we will develop several aspects of HDL metabolism with emphasis on the structure/function of apo A-I and apo A-II. An important contribution to our understanding of the respective roles of apo A-I and apo A-II comes from studies using transgenic animal models that highlighted the stabilizatory role of apo A-II on HDL through inhibition of their remodeling by lipases. Clinical studies coupled with proteomic analyses revealed the presence of dysfunctional HDL in patients with cardiovascular disease. Beyond HDL cholesterol, a new notion is the functionality of HDL particles. In spite of abundant literature on HDL metabolic properties, a major question remains unanswered: which HDL particle(s) confer(s) protection against cardiovascular risk?

The acquired coagulationfactor inhibitors are classified into alloantibodies, which appear in association with supplementary treatment for congenital coagulationfactor deficiency, and autoantibodies, which are spontaneously produced. We report here 2 cases of acquired factor VIII inhibitor and 1 case of factor V inhibitor. Case 1: A 52-year-old woman noted swelling of the right parotid region in March 1988. Though contrast examination was scheduled, she was admitted for detailed examination due to a markedly prolonged coagulation time. An APTT correction test suggested that decreased factor VIII activity was due to the presence of an inhibitor. Since antinuclear antibody and SS-A antibody were positive and infiltration by lymphocytes in the salivary gland acini in a lip biopsy specimen was detected, Sjögren's syndrome was diagnosed. Case 2: A 33-year-old woman had normal delivery of her second child in February 1998. In June 1998, she suffered slight contusion in the left lower limb. The affected site became swollen and painful, making walking difficult. Since both upper limbs became markedly swollen after 1 week, she visited our hospital. Prolonged APTT and a marked decrease in factor VIII activity were observed. Factor VIII inhibitor titer was high at 19 Bethesda units. Case 3: A 64-year-old man had had asymptomatic macroscopic hematuria since the beginning of August 1998 but was placed under observation since no abnormal findings were observed on various imaging tests. However, he was admitted to Osaka City General Medical Center because of vesicular tamponade. Factor V activity was markedly decreased to 1.0%. PT correction test suggested that decreased factor V activity was due to the presence of an inhibitor. The underlying disease could not be determined in this case. In patients with acquired coagulation inhibitors, bleeding symptoms are reported to be mild in many cases, and severe bleeding is rare. However, cases of death without severe bleeding or

The hypotension and disseminated intravascular coagulation (DIC) in bacteremia is thought to be mediated by the combined actions of cytokines, prostaglandins, and complement. The contact system, via the release of bradykinin and the activation of Factor XI, has been postulated to be contributing to the observed hypotension and DIC. Using a mAb to Factor XII (C6B7), we blocked the activation of the contact system in an established experimental baboon model in which Escherichia coli was infused to produce lethal bacteremia with hypotension. The untreated group (n = 5) displayed contact activation, manifested by a significant decrease in high molecular weight kininogen (HK) and a significant increase in alpha 2 macroglobulin-kallikrein complexes (alpha 2M-Kal). The C6B7-treated group (n = 5) showed an inactivation of Factor XII and the changes in HK and alpha 2M-Kal complexes were prevented. Both groups developed DIC manifested by a decrease in platelet, fibrinogen, and Factor V levels. The untreated group developed irreversible hypotension. The treated group experienced an initial hypotension that was reversed and extended the life of the animals. This study suggests that irreversible hypotension correlates with prolonged activation of the contact system, and specific antibody therapy can modulate both the pathophysiological and biochemical changes. Images PMID:7678610

Acute myocardial infarction is the first cause of morbidity and mortality in the world, resulting in the combination of genetic and environmental factors. It has been postulated that the R353Q polymorphism of the coagulation FVII gene represents a protective factor for acute myocardial infarction, whereas the N700S polymorphism in the thrombospondin-1 gene is associated with an increased risk for acute myocardial infarction; however, the results are still contradicted. The objective of the study was to examine the possible association of the FVII R353Q and N700S polymorphism and acute myocardial infarction in Mexican patients with acute myocardial infarction younger than 45 years old. Case-control study that included 252 patients who were diagnosed with acute myocardial infarction and 252 apparently healthy, age- and gender-matched individuals without a history of coronary artery disease. R353Q and N700S polymorphisms were determined in all participants by PCR-RFLP. There was no statistical significant difference in genotype distribution (p = 0.06) between the acute myocardial infarction and control groups. Also, there was a similar genotype distribution of N700S polymorphism between stroke and control groups (p = 0.50). Hypertension, diabetes mellitus, family history of coronary disease and dyslipidemia represented independent risk factors for acute myocardial infarction. Polymorphisms R353Q and N700S do not represent a protective or risk factor for acute myocardial infarction in young Mexican individuals.

Several studies have suggested a role for blood coagulation proteins in tumour progression. Herein, we discuss (1) the activation of the blood clotting cascade in the tumour microenvironment and its impact on primary tumour growth; (2) the intravascular activation of blood coagulation and its impact on tumour metastasis and cancer-associated thrombosis; and (3) antitumour therapies that target blood-coagulation-associated proteins. Expression levels of the clotting initiator protein TF (tissue factor) have been correlated with tumour cell aggressiveness. Simultaneous TF expression and PS (phosphatidylserine) exposure by tumour cells promote the extravascular activation of blood coagulation. The generation of blood coagulation enzymes in the tumour microenvironment may trigger the activation of PARs (protease-activated receptors). In particular, PAR1 and PAR2 have been associated with many aspects of tumour biology. The procoagulant activity of circulating tumour cells favours metastasis, whereas the release of TF-bearing MVs (microvesicles) into the circulation has been correlated with cancer-associated thrombosis. Given the role of coagulation proteins in tumour progression, it has been proposed that they could be targets for the development of new antitumour therapies. PMID:23889169

Several studies have suggested a role for blood coagulation proteins in tumour progression. Herein, we discuss (1) the activation of the blood clotting cascade in the tumour microenvironment and its impact on primary tumour growth; (2) the intravascular activation of blood coagulation and its impact on tumour metastasis and cancer-associated thrombosis; and (3) antitumour therapies that target blood-coagulation-associated proteins. Expression levels of the clotting initiator protein TF (tissue factor) have been correlated with tumour cell aggressiveness. Simultaneous TF expression and PS (phosphatidylserine) exposure by tumour cells promote the extravascular activation of blood coagulation. The generation of blood coagulation enzymes in the tumour microenvironment may trigger the activation of PARs (protease-activated receptors). In particular, PAR1 and PAR2 have been associated with many aspects of tumour biology. The procoagulant activity of circulating tumour cells favours metastasis, whereas the release of TF-bearing MVs (microvesicles) into the circulation has been correlated with cancer-associated thrombosis. Given the role of coagulation proteins in tumour progression, it has been proposed that they could be targets for the development of new antitumour therapies.

Disorders of coagulation in children often prove challenging to the medical care team. The aims of this study were to assess the spectrum and prevalence of coagulation disorders among children attending Mansoura University Children Hospital (MUCH), Mansoura, Egypt. A total of 105 pediatric patients were referred to MUCH. They were divided into two groups: congenital coagulation disorders (75 cases, age 45.36 +/- 48.59 months), and acquired coagulation disorders (30 cases, age 56.13 +/- 61.61 months). All patients were subjected to thorough history taking including the nature of bleeding, family, past history, mode of inheritance, and detailed physical findings. Hemostatic tests included: platelet count, bleeding time (BT), prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT). Specific tests in the congenital group include assay of coagulationfactors according to each disorder, Von Willebrand factor assay, ristocetin aggregation test, APTT mixing study for detection of inhibitors in complicated hemophilia cases, F VIII C to VWAg ratio with cut off 0.7 for detection of carriers in some hemophilia A families. Congenital disorders constituted 71.4% of the studied cases vs. 28.6% for acquired disorders. Hemophilia A (42.85%), hemophilia B (14.28%) and liver diseases (14.28%) represented the majority of the studied cases. Mild and moderate cases of hemophilia A and B are more frequent than severe cases in both types. Male sex is more frequent than female in the congenital group (94.7 vs. 5.3%, P < 0.001). Direct correlation existed between factor level assay and severity of hemophilia (r = 0.73, P = 0.006). Three mothers and one sister were identified as carrier out of four families. Anti-clotting factors inhibitor was detected in 18.2% of patients with hemophilia A and in 9.1% with hemophilia B. In conclusion, our study found that hemophilias are the most prevalent congenital coagulation disorders among children. Attention must be

A new inorganic-organic hybrid material zirconium-glycine complex (ZGC) was firstly used as a coagulant in a coagulation process to treat Pearl River raw water. Its coagulation performance was compared with commonly used aluminum (Al) coagulants such as aluminum sulfate (Al2(SO4)3) and polyaluminum chloride (PAC), in terms of water quality parameters and floc properties. ZGC coagulation achieved higher removal of turbidity (93.8%) than other traditional coagulants. Charge neutralization was proven to act as a dominant mechanism during ZGC coagulation. The aggregated flocs with ZGC showed the fastest growth rate and good recovery ability compared with the other coagulants and achieved the largest floc size within 5 min. The ZGC coagulant can decrease the hydraulic retention time and increase removal efficiency.

In physico-chemical treatment, the separation of suspended particles from the liquid phase is usually accomplished by coagulation, flocculation and sedimentation. Coagulation-flocculation processes have been widely used as alternative treatment to remove leachate pollutants such as BOD, COD, TSS, heavy metals, colour, and nitrogen compounds prior to other treatment methods. It is often coupled with treatment methods like biological process, chemical oxidation, adsorption or filtration to achieve desirable effluent quality. In spite of being economical, the dewatering and disposal of the precipitated sludge could be laborious and time-consuming. In this manuscript, brief discussions on coagulant and coagulants aid in landfill leachate treatment is discussed with respect to their mechanism.

Blood coagulation time is an important factor to consider for postoperative and cardiac disorder patients who have been prescribed anticoagulant coagulant medications. The coagulation process is also known to be perturbed in some individuals with psychiatric disorders, such as schizophrenia. This chapter describes a patient self-management system for a functional assessment of blood coagulation activity, determining the appropriate anticoagulant dosages using a test strip